Group B Streptococcus polypeptides nucleic acids and therapeutic compositions and vaccines thereof

ABSTRACT

This invention provides isolated nucleic acids encoding polypeptides comprising amino acid sequences of streptococcal matrix adhesion (Ema) polypeptides. The invention provides nucleic acids encoding Group B streptococcal Ema polypeptides EmaA, EmaB, EmaC, EmaD and EmaE. The present invention provides isolated polypeptides comprising amino acid sequences of Group B streptococcal polypeptides EmaA, EmaB, EmaC, EmaD and EmaE, including analogs, variants, mutants, derivatives and fragments thereof. Ema homologous polypeptides from additional bacterial species, including  S. pneumoniae, S. pyogenes, E. faecalis  and  C. diptheriae  are also provided. Antibodies to the Ema polypeptides and immunogenic fragments thereof are also provided. The present invention relates to the identification and prevention of infections by virulent forms of streptococci. This invention provides pharmaceutical compositions, immunogenic compositions, vaccines, and diagnostic and therapeutic methods of use of the isolated polypeptides, antibodies thereto, and nucleic acids. Assays for compounds which modulate the polypeptides of the present invention for use in therapy are also provided.

This application is a divisional of U.S. patent application Ser. No.11/493,705 filed Jul. 26, 2006, now U.S. Pat. No. 7,645,577, which is acontinuation application of U.S. patent application Ser. No. 10/333,002,filed Jul. 8, 2003, now U.S. Pat. No. 7,128,919, which is a U.S.National Stage application of PCT/US2001/024795 filed Aug. 8, 2001,which claims the benefit of priority to U.S. application Ser. No.09/634,341, filed Aug. 8, 2000, each of which are herein incorporated byreference in their entirety.

The research leading to the present invention was supported, at least inpart, by a grant from NAID, Grant No. A140918. Accordingly, theGovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to extracellular matrix adhesin (Ema)proteins, antibodies thereto and to vaccines, compositions andtherapeutics. The Group B streptococcal Ema polypeptides are EmaA, EmaB,EmaC, EmaD and EmaE. The invention further relates to Ema polypeptidesfrom various species of bacteria, including S. pneumoniae, S. pyogenes,E. faecalis and C. diptheriae. The invention also relates to theidentification and prevention of infections by streptococci. Isolatednucleic acids encoding Group B streptococcal Ema polypeptides,particularly EmaA, EmaB, EmaC, EmaD and EmaE and to other bacterial Emahomologs are included herein. Assays for compounds which modulate thepolypeptides of the present invention for use in therapy are alsoprovided.

BACKGROUND OF THE INVENTION

Streptococci are catalase negative gram positive cocci. They may beclassified by the type of hemolysis exhibited on blood agar, by theserologic detection of carbohydrate antigens, or by certain biochemicalreactions. Medically important streptococci include Groups A, B, D, S.pneumoniae and the viridans group of streptococci. Lancefield type A(GroupA) Streptococcus pyogenes is an important human pathogen—the causeof streptococcal pharyngitis, impetigo and more severe infections suchas bacterernia and necrotizing fascitis. The immunologic sequelae ofGroup A Streptococcal infections are also important healthproblems—rheumatic carditis is the most common cause of acquired cardiacdisease worldwide and post-streptococcal glomerulonephritis is a causeof hypertension and renal dysfunction. Group B Streptococcus agalactiaeare the most common cause of serious bacterial infections in newborns,and important pathogens in pregnant women and nonpregnant adults withunderlying medical problems such as diabetes and cardiovascular disease.Group D streptococci include the enterococci (Streptococcus faecalis andfaecium) and the “nonenterococcal” Group D streptococci. Streptococcuspneumoniae (pneumococcus) is not classified by group in the Lancefieldsystem. Pneumococci are extremely important human pathogens, the mostcommon cause of bacterial pneumonia, middle ear infections andmeningitis beyond the newborn period. The viridans group of streptococciinclude S. milleri, S. mitis, S. sanguis and others. They causebacteremia, endocarditis, and dental infections. Enterococci areimportant causes of urinary tract infections, bacteremia and woundinfections (predominantly as nosocomial infections in hospitalizedpatients), and endocarditis. Over the past decade enterococci havedeveloped resistance to many conventional antibiotics and there are somestrains resistant to all known antibiotics.

Group B streptococci (GBS) are the most common cause of seriousbacterial disease in neonates, and are important pathogens in pregnantwomen and adults with underlying illnesses (Baker C J. (2000) “Group Bstreptococcal infections” in Streptococcal infections. Clinical aspects,microbiology, and molecular pathogenesis. (D. L. Stevens and E. L.Kaplan), New York: Oxford University Press, 222-237). Commonmanifestations of these infections include bacteremia, pneumonia,meningitis, endocarditis, and osteoarticular infections (Baker C J.(2000) “Group B streptococcal infections” in Streptococcal infections.Clinical aspects, microbiology, and molecular pathogenesis. (D. L.Stevens and E. L. Kaplan), New York: Oxford University Press, 222-237;Blumberg H. M. et al. (1996) J Infect Dis 173:365-373). The incidence ofinvasive GBS disease is approximately 2.6 in 1000 live births and 7.7 in100,000 in the overall population, with mortality rates that vary from 6to 30% (Baker C J. (2000) “Group B streptococcal infections” inStreptococcal infections. Clinical aspects, microbiology, and molecularpathogenesis. (D. L. Stevens and E. L. Kaplan), New York: OxfordUniversity Press, 222-237; Blumberg H. M. et al. (1996) J Infect Dis173:365-373). Although much neonatal disease is preventable byadministration of prophylactic antibiotics to women in labor, antibioticprophylaxis programs can be inefficient, suffer from poor compliance, orfail if antibiotic resistance emerges. No effective prophylaxis strategyfor adult infections has been established.

During childbirth, GBS can pass from the mother to the newborn. By oneestimate, up to 30% of pregnant women carry GBS at least temporarily inthe vagina or rectum without symptoms. Infants born to these womenbecome colonized with GBS during delivery (Baker, C. J. and Edwards, M.S. (1995) “Group B Streptococcal Infections” in Infectious Disease ofthe Fetus and Newborn Infant (J. S. Remington and J. O Klein),980-1054). Aspiration of infected amniotic fluid or vaginal secretionsallow GBS to gain access to the lungs. Adhesion to, and invasion of,respiratory epithelium and endothelium appear to be critical factors inearly onset neonatal infection. (Baker, C. J. and Edwards, M. S. (1995)“Group B Streptococcal Infections” in Infectious Disease of the Fetusand Newborn Infant (J. S. Remington and J. O Klein), 980-1054; Rubens,C. E. et al. (1991) J Inf Dis 164:320-330). Subsequent steps ininfection, such as blood stream invasion and the establishment ofmetastatic local infections have not been clarified. The pathogenesis ofneonatal infection occurring after the first week of life is also notwell understood. Gastrointestinal colonization may be more importantthan a respiratory focus in late onset neonatal disease (Baker, C. J.and Edwards, M. S. (1995) “Group B Streptococcal Infections” inInfectious Disease of the Fetus and Newborn Infant (J. S. Remington andJ. O Klein), 980-1054). Considerable evidence suggests that invasion ofbrain microvascular endothelial cells by GBS is the initial step in thepathogenesis of meningitis. GBS are able to invade human brainmicrovascular endothelial cells and type III GBS, which are responsiblefor the majority of meningitis, accomplish this 2-6 times moreefficiently than other serotypes (Nizet, V. et al. (1997) Infect Immun65:5074-5081).

Because GBS is widely distributed among the population and is animportant pathogen in newborns, pregnant women are commonly tested forGBS at 35-37 weeks of pregnancy. Much of GBS neonatal disease ispreventable by administration of prophylactic antibiotics during laborto women who test positive or display known risk factors. However, theseantibiotics programs do not prevent all GBS disease. The programs aredeficient for a number of reasons. First, the programs can beinefficient. Second, it is difficult to ensure that all healthcareproviders and patients comply with the testing and treatment. Andfinally, if new serotypes or antibiotic resistance emerges, theantibiotic programs may fail altogether. Currently available tests forGBS are inefficient. These tests may provide false negatives.Furthermore, the tests are not specific to virulent strains of GBS.Thus, antibiotic treatment may be given unnecessarily and add to theproblem of antibiotic resistance. Although a vaccine would beadvantageous, none are yet commercially available.

Traditionally, GBS are divided into 9 serotypes according to theimmunologic reactivity of the polysaccharide capsule (Baker C J. (2000)“Group B streptococcal infections” in Streptococcal infections. Clinicalaspects, microbiology, and molecular pathogenesis. (D. L. Stevens and E.L. Kaplan), New York: Oxford University Press, 222-237; Blumberg H. M.et al. (1996) J Infect Dis 173:365-373; Kogan, G. et al. (1996) J BiolChem 271:8786-8790). Serotype III GBS are particularly important inhuman neonates, causing 60-70% of all infections and almost allmeningitis (Baker C J. (2000) “Group B streptococcal infections” inStreptococcal infections Clinical aspects, microbiology, and molecularpathogenesis. (D. L. Stevens and E. L. Kaplan), New York: OxfordUniversity Press, 222-237). Type III GBS can be subdivided into threegroups of related strains based on the analysis of restriction digestpatterns (RDPs) produced by digestion of chromosomal DNA with Hind IIIand Sse8387. (I. Y. Nagano et al. (1991) J Med Micro 35:297-303; S.Takahashi et al. (1998) J Inf Dis 177:1116-1119).

Over 90% of invasive type III GBS neonatal disease in Tokyo, Japan andin Salt Lake City, Utah is caused by bacteria from one of three RDPtypes, termed RDP type III-3, while RDP type III-2 are significantlymore likely to be isolated from vagina than from blood or CSF. Theseresults suggest that this genetically-related cluster of type III-3 GBSare more virulent than III-2 strains and could be responsible for themajority of invasive type III disease globally.

Preliminary vaccines for GBS used unconjugated purified polysaccaride.GBS poly- and oligosaccharides are poorly immunogenic and fail to elicitsignificant memory and booster responses. Baker et al immunized 40pregnant women with purified serotype III capsular polysaccharide(Baker, C. J. et al. (1998) New Engl J of Med 319:1180-1185). Overall,only 57% of women with low levels of specific antibody responded to thevaccine. The poor immunogenicity of purified polysaccharide antigen wasfurther demonstrated in a study in which thirty adult volunteers wereimmunized with a tetravalent vaccine composed of purified polysaccharidefrom serotypes Ia, Ib, II, and III (Kotloff, K. L. et al. (1996) Vaccine14:446-450). Although safe, this vaccine was only modestly immunogenic,with only 13% of subjects responding to type Ib, 17% to type II, 33%responding to type Ia, and 70% responding to type III polysaccharide.The poor immunogenicity of polysaccharide antigens prompted efforts todevelop polysaccharide conjugate vaccines, whereby these poly- oroligosaccharides are conjugated to protein carriers. Ninety percent ofhealthy adult women immunized with a type III polysaccharide-tetanustoxoid conjugate vaccine responded with a 4-fold rise in antibodyconcentration, compared to 50% immunized with plain polysaccharide(Kasper, D. L. et al (1996) J of Clin Invest 98:2308-2314). A type Ia/Ibpolysaccharide-tetanus toxoid conjugate vaccine was similarly moreimmunogenic in healthy adults than plain polysaccharide (Baker, C. J. etal (1999) J Infect Dis 179:142-150).

The disadvantage of polysaccharide-protein conjugate vaccines is thatthe process of purifying and conjugating polysaccharides is difficult,time-consuming and expensive. A protein antigen which could be cheaplyand easily produced would be an improvement.

If one were to make a polysaccharide-protein conjugate vaccine, aGBS-specific carrier protein may be preferable to one of the commonlyused carriers such as tetanus or diphtheria toxoids because of thepotential problems associated with some of these carrier proteins,particularly variable immunogenicity and the problems associated withrepeated vaccination with the same carrier protein. Selection ofappropriate carrier proteins is important for the development ofpolysaccharide-protein vaccine formulations. For example, Haemophilusinfluenzae type b poly- or oligosaccharide conjugated to differentprotein carriers has variable immunogenicity and elicits antibody withvarying avidity (Decker, M. D. et al (1992) J Pediatrics 120:184-189;Schlesinger, Y. (1992) JAMA 267:1489-1494). Repeated immunization withthe same carrier protein may also suppress immune responses bycompetition for specific B cells (epitopic suppression) or othermechanisms. This is of particular concern for the development of GBSvaccines since recently developed poly/oligosaccharide-protein conjugatevaccines against the bacteria H. influenzae, S. pneumoniae, and N.meningitidis all utilize a restricted number of carrier proteins(tetanus toxoid, CRM197, diptheria toxoid), increasing the number ofexposures to these carriers an individual is likely to receive.Additionally, using tetanus as a carrier protein offers no specificadvantage beyond the improved immunogenicity of the vaccine. Asecond-generation vaccine containing a GBS-specific carrier proteinwould enhance immunogenicity and have an advantage in that aGBS-specific immune response would be generated against both the carrierprotein and the poly/oligosaccharide.

Therefore, in view of the aforementioned deficiencies attendant withprior art vaccines and methods, it should be apparent that there stillexists a need in the art for an effective and immunogenic GBS vaccine.The availability and use of a GBS polypeptide in a conjugate vaccine isdesirable. A GBS polypeptide which is present or expressed in all GBSserotypes would have the added advantage of providing broad, generalimmunity across many GBS serotypes. It would be particularly relevantand useful to provide a streptococcal vaccine or immunogen which isexpressed broadly in various streptococcal species, whereby broad orgeneral immunity against multiple and unique groups of streptococci (forinstance, Group A, Group B and S. pneumoniae), particularly againstdistinct virulent and clinically relevant streptococcal bacteria, couldthereby be generated.

The citation of references herein shall not be construed as an admissionthat such is prior art to the present invention.

SUMMARY OF THE INVENTION

In accordance with the present invention, streptococcal polypeptidestermed extracellular matrix adhesins (Ema) are provided which areparticularly useful in the identification and prevention of infectionsby streptococci.

In its broadest aspect, the present invention encompasses isolatedpolypeptides comprising an amino acid sequence of a streptococcalpolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.The isolated peptides, including combinations of one or more thereof,are suitable for use in immunizing animals and humans against bacterialinfection, particularly streptococci.

The present invention is directed to an isolated streptococcal EmaApolypeptide which comprises the amino acid sequence set out in SEQ IDNO: 2, and analogs, variants and immunogenic fragments thereof.

The present invention is directed to an isolated streptococcal EmaBpolypeptide which comprises the amino acid sequence set out in SEQ IDNO: 4, and analogs, variants and immunogenic fragments thereof.

The present invention is directed to an isolated streptococcal EmaCpolypeptide which comprises the amino acid sequence set out in SEQ IDNO: 6, and analogs, variants and immunogenic fragments thereof.

The present invention is directed to an isolated streptococcal EmaDpolypeptide which comprises the amino acid sequence set out in SEQ IDNO: 8, and analogs, variants and immunogenic fragments thereof.

The present invention is directed to an isolated streptococcal EmaEpolypeptide which comprises the amino acid sequence set out in SEQ IDNO: 10, and analogs, variants and immunogenic fragments thereof.

The present invention also provides Ema polypeptide homologs fromdistinct bacterial species, particularly including distinctstreptococcal species, more particularly including Group Bstreptococcus, Group A streptococcus (particularly S. pyogenes) and S.pneumoniae. The present invention also provides Ema polypeptides fromadditional distinct bacterial species, particularly includingEnterococcus faecalis and Corynebacterium diptheriae. Nucleic acidsencoding Ema polypeptide homologs from distinct bacterial species arealso provided.

The present invention thus provides an isolated streptococcal Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO:23.An isolated nucleic acid which encodes the streptococcal polypeptide setout in SEQ ID NO:23 is further provided.

The invention thus further provides an isolated streptococcal Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO:26.An isolated nucleic acid which encodes the streptococcal polypeptide setout in SEQ ID NO:26 is further provided.

The present invention further provides an isolated streptococcal Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO:37.An isolated nucleic acid which encodes the streptococcal polypeptide setout in SEQ ID NO:37 is further provided.

An enterococcal Ema polypeptide is further provided comprising the aminoacid sequence set out in SEQ ID NO:29. An isolated nucleic acid whichencodes the enterococcal polypeptide set out in SEQ ED NO:29 is alsoprovided.

The invention provides an isolated Corynebacterium Ema polypeptidecomprising the amino acid sequence set out in SEQ ID NO: 32. Alsoprovided is an isolated nucleic acid which encodes the Corynebacteriumpolypeptide set out in SEQ ID NO: 32.

The invention provides an isolated bacterial polypeptide comprising theamino acid sequence TLLTCTPYMINS/THRLLVR/KG (SEQ ID NO: 34), wherein thepolypeptide is not isolated from Actinomyces.

The invention further provides an isolated streptococcal polypeptidecomprising the amino acid sequence TLLTCTPYMINS/THRLLVR/KG (SEQ ID NO:34).

Also provided is an isolated bacterial polypeptide comprising the aminoacid sequence TABLE-US-00001 TLVTCTPYGINTHRLLVTA. (SEQ ID NO: 35)

The present invention includes an isolated bacterial polypeptidecomprising the amino acid sequence TLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36).An isolated streptococcal polypeptide comprising the amino acid sequenceTLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36) is also provided.

The invention further includes an isolated polypeptide having the aminoacid sequence selected from the group of TLLTCTPYMINS/THRLLVR/KG (SEQ IDNO: 34), TLVTCTPYGINTHRLLVTA (SEQ ID NO: 35), and TLVTCTPYGVNTKRLLVRG(SEQ ID NO: 36).

The present invention contemplates the use of the polypeptides of thepresent invention in diagnostic tests and methods for determining and/ormonitoring of streptococcal infection. Thus, the present inventionprovides an isolated Ema polypeptide, particularly selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE, labeled with a detectablelabel.

In the instance where a radioactive label, such as the isotopes .sup.3H,sup.14C, .sup.32P, .sup.35S, .sup.36Cl, .sup.51Cr, .sup.57Co, .sup.58Co,.sup.59Fe, .sup.90Y, .sup.125I, .sup.131I, and .sup.186Re are used,known currently available counting procedures may be utilized. In theinstance where the label is an enzyme, detection may be accomplished byany of the presently utilized calorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques known inthe art.

The present invention extends to an immunogenic Ema polypeptide,particularly selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,or a fragment thereof. The present invention also extends to immunogenicEma polypeptides wherein such polypeptides comprise a combination of atleast one immunogenic Ema polypeptide, selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE, or immunogenic polypeptide fragment thereof,and a GBS polypeptide selected from the group of Spb1, Spb2, C proteinalpha antigen, Rib, Lmb, C5a-ase, or immunogenic fragments thereof.

In a further aspect, the present invention extends to vaccines based onthe Ema proteins described herein. The present invention provides avaccine comprising one or more streptococcal polypeptides selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, and a pharmaceuticallyacceptable adjuvant. The present invention provides a vaccine comprisingone or more streptococcal polypeptides selected from the group of thepolypeptide of SEQ ID NO: 23, 26, and 37, and a pharmaceuticallyacceptable adjuvant.

The present invention further provides a streptococcal vaccinecomprising one or more Group B streptococcal polypeptides selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, further comprising one ormore additional streptococcal antigens. The present invention furtherprovides a GBS vaccine comprising one or more Group

B streptococcal polypeptides selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE, further comprising one or more additional GBSantigens. In a particular embodiment, the GBS antigen is selected fromthe group of the polypeptide Spb1 or an immunogenic fragment thereof,the polypeptide Spb2 or an immunogenic fragment thereof, C protein alphaantigen or an immunogenic fragment thereof, Rib or an immunogenicfragment thereof. Lmb or an immunogenic fragment thereof, C5a-ase or animmunogenic fragment thereof and Group B streptococcal polysaccharidesor oligosaccharides.

In another aspect, the invention is directed to a vaccine for protectionof an animal subject from infection with streptococci comprising animmunogenic amount of one or more Etma polypeptide EmaA, EmaB, EmaC,EmaD or EmaE, or a derivative or fragment thereof. Such a vaccine maycontain the protein conjugated covalently to a GBS bacterialpolysaccharide or oligosaccharide or polysaccharide or oligosaccharidefrom one or more GBS serotypes.

In a still further aspect, the present invention provides an immunogeniccomposition comprising one of more streptococcal polypeptides selectedfrom the group of EmaA, EmaB, EmaC, EmaD and EmaE, and apharmaceutically acceptable adjuvant.

The present invention further provides an immunogenic compositioncomprising one or more Group B streptococcal polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, further comprising one ormore antigens selected from the group of the polypeptide Spb1 or animmunogenic fragment thereof, the polypeptide Spb2 or an immunogenicfragment thereof, C protein alpha antigen or an immunogenic fragmentthereof, Rib or an immunogenic fragment thereof. Lmb or an immunogenicfragment thereof, C5a-ase or an immunogenic fragment thereof, and GroupB streptococcal polysaccharides or oligosaccharides.

The invention further provides pharmaceutical compositions, vaccines,and diagnostic and therapeutic methods of use thereof.

The invention provides pharmaceutical compositions comprising abacterial Ema polypeptide and a pharmaceutically acceptable carrier. Theinvention provides pharmaceutical compositions comprising astreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE, the polypeptide of SEQ ID NO:23, the polypeptide of SEQID NO: 26, the polypeptide of SEQ ID NO:37, and a pharmaceuticallyacceptable carrier. The invention provides pharmaceutical compositionscomprising a streptococcal polypeptide selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE, and a pharmaceutically acceptable carrier.The present invention further provides pharmaceutical compositionscomprising one or more GBS Ema polypeptide, or a fragment thereof, incombination with one or more of GBS polypeptide Spb1, Spb2, C proteinalpha antigen, Rib, Lmb, C5a-ase, a Group B streptococcal polysaccharideor oligosaccharide vaccine, and an anti-streptococcal vaccine.

In a still further aspect, the present invention provides a purifiedantibody to a streptococcal polypeptide selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE. In a still further aspect, the presentinvention provides a purified antibody to a streptococcal polypeptideselected from the group of the polypeptide of SEQ ID NO:23, thepolypeptide of SEQ ID NO: 26, and the polypeptide of SEQ ID NO:37.

Antibodies against the isolated polypeptides of the present inventioninclude naturally raised and recombinantly prepared antibodies. Thesemay include both polyclonal and monoclonal antibodies prepared by knowngenetic techniques, as well as bi-specific (chimeric) antibodies, andantibodies including other functionalities suiting them for diagnosticuse. Such antibodies can be used in immunoassays to diagnose infectionwith a particular strain or species of bacteria. The antibodies can alsobe used for passive immunization to treat an infection withstreptococcal bacteria including Group B streptococcus, Group Astreptococcus, and S. pneumoniae. These antibodies may also be suitablefor modulating bacterial adherence and/or invasion including but notlimited to acting as competitive agents.

The present invention provides a monoclonal antibody to a streptococcalpolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.The invention thereby extends to an immortal cell line that produces amonoclonal antibody to a streptococcal polypeptide selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE.

An antibody to a streptococcal Ema polypeptide EmaA, EmaB, EmaC, EmaD orEmaE labeled with a detectable label is further provided. In particularembodiments, the label may selected from the group consisting of anenzyme, a chemical which fluoresces, and a radioactive element.

The present invention provides a pharmaceutical composition comprisingone or more antibodies to a streptococcal protein selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE, and a pharmaceuticallyacceptable carrier. The invention further provides a pharmaceuticalcomposition comprising a combination of at least two antibodies to GroupB streptococcal proteins and a pharmaceutically acceptable carrier,wherein at least one antibody to a protein selected from the group ofEmaA, EmaB, EmaC, EmaD, and EmaE is combined with at least one antibodyto a protein selected from the group of Spb1, Spb2, Rib, Lmb, C5a-aseand a C protein alpha antigen.

The present invention also relates to isolated nucleic acids, such asrecombinant DNA molecules or cloned genes, or degenerate variantsthereof, mutants, analogs, or fragments thereof, which encode theisolated polypeptide of the present invention or which competitivelyinhibit the activity of the polypeptide. The present invention furtherrelates to isolated nucleic acids, such as recombinant DNA molecules orcloned genes, or degenerate variants thereof, mutants, analogs, orfragments thereof, which encode a bacterial Ema polypeptide. The presentinvention further relates to isolated nucleic acids, such as recombinantDNA molecules or cloned genes, or degenerate variants thereof, mutants,analogs, or fragments thereof, which encode a streptococcal Emapolypeptide. The present invention further relates to isolated nucleicacids, such as recombinant DNA molecules or cloned genes, or degeneratevariants thereof, mutants, analogs, or fragments thereof, which encode astreptococcal Ema polypeptide, particularly selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE. Preferably, the isolated nucleic acid,which includes degenerates, variants, mutants, analogs, or fragmentsthereof, has a sequence as set forth in SEQ ID NOS: 1, 3, 5, 7 or 9. Ina further embodiment of the invention, the DNA sequence of therecombinant DNA molecule or cloned gene may be operatively linked to anexpression control sequence which may be introduced into an appropriatehost. The invention accordingly extends to unicellular hosts transformedwith the cloned gene or recombinant DNA molecule comprising a DNAsequence encoding an Ema protein, particularly selected from the groupof EmaA, EmaB, EmaC, EmaD and EmaE, and more particularly, the DNAsequences or fragments thereof determined from the sequences set forthabove.

In a particular embodiment, the nucleic acid encoding the EmaApolypeptide has the sequence selected from the group comprising SEQ IDNO:1; a sequence that hybridizes to SEQ ID NO:1 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:1 or a sequence thathybridizes to SEQ ID NO:1 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaA polypeptide has the sequence selected from thegroup comprising SEQ ID NO:1; a sequence complementary to SEQ ID NO:1;or a homologous sequence which is substantially similar to SEQ ID NO:1.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:1.

In a particular embodiment, the nucleic acid encoding the EmaBpolypeptide has the sequence selected from the group comprising SEQ IDNO:3; a sequence that hybridizes to SEQ ID NO:3 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:3 or a sequence thathybridizes to SEQ ID NO:3 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaB polypeptide has the sequence selected from thegroup comprising SEQ ID NO:3; a sequence complementary to SEQ ID NO:3;or a homologous sequence which is substantially similar to SEQ ID NO:3.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:3.

In a particular embodiment, the nucleic acid encoding the EmaCpolypeptide has the sequence selected from the group comprising SEQ IDNO:5; a sequence that hybridizes to SEQ ID NO:5 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:5 or a sequence thathybridizes to SEQ ID NO:5 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaC polypeptide has the sequence selected from thegroup comprising SEQ ID NO:5; a sequence complementary to SEQ ID NO:5;or a homologous sequence which is substantially similar to SEQ ID NO:5.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:5.

In a particular embodiment, the nucleic acid encoding the EmaDpolypeptide has the sequence selected from the group comprising SEQ IDNO:7; a sequence that hybridizes to SEQ ID NO:7 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:7 or a sequence thathybridizes to SEQ ID NO:7 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaD polypeptide has the sequence selected from thegroup comprising SEQ ID NO:7; a sequence complementary to SEQ ID NO:7;or a homologous sequence which is substantially similar to SEQ ID NO:7.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:7.

In a particular embodiment, the nucleic acid encoding the EmaEpolypeptide has the sequence selected from the group comprising SEQ IDNO:9; a sequence that hybridizes to SEQ ID NO:9 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:9 or a sequence thathybridizes to SEQ ID NO:9 under moderate stringency hybridizationconditions; degenerate variants thereof, alleles thereof; andhybridizable fragments thereof In a particular embodiment, the nucleicacid encoding the EmaE polypeptide has the sequence selected from thegroup comprising SEQ ID NO:9; a sequence complementary to SEQ ID NO:9;or a homologous sequence which is substantially similar to SEQ ID NO:9In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:9.

In a further embodiment, the nucleic acid encoding the bacterial Emapolypeptide comprises the sequence selected from the group comprisingSEQ ID NO: 24, 27, 30 and 33. In a further embodiment, the nucleic acidencoding the bacterial Ema polypeptide has the sequence selected fromthe group comprising SEQ ID NO: 24, 27, 30 and 33.

A nucleic acid capable of encoding a streptococcal polypeptide EmaA,EmaB, EmaC, EmaD or EmaE which is a recombinant DNA molecule is furtherprovided. Such a recombinant DNA molecule wherein the DNA molecule isoperatively linked to an expression control sequence is also providedherein.

The present invention relates to nucleic acid vaccines or DNA vaccinescomprising nucleic acids encoding immunogenic streptococcal Emapolypeptides, particularly selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE. The present invention relates to nucleic acid vaccines orDNA vaccines comprising nucleic acids encoding one or more immunogenicEma polypeptide or a fragment thereof or any combination of one or moreEma polypeptide EmaA, EmaB, EmaC, EmaD or EmaE with at least one otherpolypeptide, particularly a GBS polypeptide, more particularly whereinsaid other GBS polypeptide is selected from the group of Spb1, Spb2, Cprotein alpha antigen, Rib, Lmb, C5a-ase, and immunogenic polypeptidefragments thereof.

The invention further relates to a vaccine for protection of an animalsubject from infection with a streptococcal bacterium comprising avector containing a gene encoding an Ema polypeptide selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE operatively associated with apromoter capable of directing expression of the gene in the subject. Thepresent invention further provides a nucleic acid vaccine comprising arecombinant DNA molecule capable of encoding a GBS polypeptide EmaA,EmaB, EmaC, EmaD or EmaE.

The invention further relates to a vaccine for protection of an animalsubject from infection with a Group B streptococcal bacterium comprisinga vector containing a gene encoding an Ema polypeptide selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE operatively associated with apromoter capable of directing expression of the gene in the subject. Thepresent invention further provides a nucleic acid vaccine comprising arecombinant DNA molecule capable of encoding a GBS polypeptide EmaA,EmaB, EmaC, EmaD or EmaE.

The present invention provides a vector which comprises the nucleic acidcapable of encoding an Ema polypeptide selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE and a promoter. The present invention providesa vector which comprises the nucleic acid of any of SEQ ID NO: 1, 3, 5,7 or 9 and a promoter. The invention contemplates a vector wherein thepromoter comprises a bacterial, yeast, insect or mammalian promoter. Theinvention contemplates a vector wherein the vector is a plasmid, cosmid,yeast artificial chromosome (YAC), bacteriophage or eukaryotic viralDNA.

The present invention further provides a host vector system for theproduction of a polypeptide which comprises the vector capable ofencoding an Ema polypeptide, particularly selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE in a suitable host cell. A host vectorsystem is provided wherein the suitable host cell comprises aprokaryotic or eukaryotic cell. A unicellular host transformed with arecombinant DNA molecule or vector capable of encoding an Emapolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaEis thereby provided.

The present invention includes methods for determining and monitoringinfection by streptococci by detecting the presence of a streptococcalpolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.In a particular such method, the streptococcal Ema polypeptide ismeasured by:

a. contacting a sample in which the presence or activity of aStreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE is suspected with an antibody to the said streptococcalpolypeptide under conditions that allow binding of the streptococcalpolypeptide to the antibody to occur; and

b. detecting whether binding has occurred between the streptococcalpolypeptide from the sample and the antibody; wherein the detection ofbinding indicates the presence or activity of the streptococcalpolypeptide in the sample.

The present invention includes methods for determining and monitoringinfection by streptococci by detecting the presence of a streptococcalpolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.In a particular such method, the streptococcal Ema polypeptide ismeasured by:

a. contacting a sample in which the presence or activity of aStreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE is suspected with an antibody to the said streptococcalpolypeptide under conditions that allow binding of the streptococcalpolypeptide to the antibody to occur; and

b. detecting whether binding has occurred between the streptococcalpolypeptide from the sample and the antibody; wherein the detection ofbinding indicates the presence or activity of the streptococcalpolypeptide in the sample.

The present invention includes methods for determining and monitoringinfection by Group B streptococci by detecting the presence of a Group Bstreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE. In a particular such method, the streptococcal Emapolypeptide is measured by:

a. contacting a sample in which the presence or activity of a Group Bstreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE is suspected with an antibody to the said Group Bstreptococcal polypeptide under conditions that allow binding of theGroup B streptococcal polypeptide to the antibody to occur; and

b. detecting whether binding has occurred between the Group Bstreptococcal polypeptide from the sample and the antibody; wherein thedetection of binding indicates the presence or activity of the Group Bstreptococcal polypeptide in the sample.

The present invention further provides a method for detecting thepresence of a bacterium having a gene encoding a streptococcalpolypeptide selected from the group of emaA, emaB, emaC, emaD and emaE,comprising:

a. contacting a sample in which the presence or activity of thebacterium is suspected with an oligonucleotide which hybridizes to astreptococcal polypeptide gene selected from the group of emaA, emaB,emaC, emaD and emaE, under conditions that allow specific hybridizationof the oligonucleotide to the gene to occur; and

b. detecting whether hybridization has occurred between theoligonucleotide and the gene; wherein the detection of hybridizationindicates that presence or activity of the bacterium in the sample.

The invention includes an assay system for screening of potentialcompounds effective to modulate the activity of a streptococcal proteinEmaA, EmaB, EmaC, EmaD or EmaE of the present invention. In oneinstance, the test compound, or an extract containing the compound,could be administered to a cellular sample expressing the particular Emaprotein to determine the compound's effect upon the activity of theprotein by comparison with a control. In a further instance the testcompound, or an extract containing the compound, could be administeredto a cellular sample expressing the Ema protein to determine thecompound's effect upon the activity of the protein, and thereby onadherence of said cellular sample to host cells, by comparison with acontrol.

It is still a further object of the present invention to provide amethod for the prevention or treatment of mammals to control the amountor activity of streptococci, so as to treat or prevent the adverseconsequences of invasive, spontaneous, or idiopathic pathologicalstates.

It is still a further object of the present invention to provide amethod for the prevention or treatment of mammals to control the amountor activity of Group B streptococci, so as to treat or prevent theadverse consequences of invasive, spontaneous, or idiopathicpathological states.

The invention provides a method for preventing infection with abacterium that expresses a streptococcal Ema polypeptide comprisingadministering an immunogenically effective dose of a vaccine comprisingan Ema polypeptide selected from the group of EmaA, EmaB, EmaC, EmaD andEmaE to a subject.

The invention further provides a method for preventing infection with abacterium that expresses a Group B streptococcal Ema polypeptidecomprising administering an immunogenically effective dose of a vaccinecomprising an Ema polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE to a subject.

The present invention is directed to a method for treating infectionwith a bacterium that expresses a streptococcal Ema polypeptidecomprising administering a therapeutically effective dose of apharmaceutical composition comprising an Ema polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, and a pharmaceuticallyacceptable carrier to a subject.

The invention further provides a method for treating infection with abacterium that expresses a streptococcal Ema polypeptide comprisingadministering a therapeutically effective dose of a pharmaceuticalcomposition comprising an antibody to an Ema polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, and a pharmaceuticallyacceptable carrier to a subject.

In a further aspect, the invention provides a method of inducing animmune response in a subject which has been exposed to or infected witha streptococcal bacterium comprising administering to the subject anamount of the pharmaceutical composition comprising an Ema polypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, and apharmaceutically acceptable carrier, thereby inducing an immuneresponse.

The invention still further provides a method for preventing infectionby a streptococcal bacterium in a subject comprising administering tothe subject an amount of a pharmaceutical composition comprising anantibody to an Ema polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE and a pharmaceutically acceptable carrier ordiluent, thereby preventing infection by a streptococcal bacterium.

In a further aspect, the invention provides a method of inducing animmune response in a subject which has been exposed to or infected witha Group B streptococcal bacterium comprising administering to thesubject an amount of the pharmaceutical composition comprising an Emapolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,and a pharmaceutically acceptable carrier, thereby inducing an immuneresponse.

The invention still further provides a method for preventing infectionby a Group B streptococcal bacterium in a subject comprisingadministering to the subject an amount of a pharmaceutical compositioncomprising an antibody to an Ema polypeptide selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE and a pharmaceutically acceptablecarrier or diluent, thereby preventing infection by a streptococcalbacterium.

The invention further provides an ema mutant bacteria which isnon-adherent and/or non-invasive to cells, particularly which is mutatedin one or more genes selected from the group of emaA, emaB, emaC, emaDand emaE. Particularly, such ema mutant is a streptococcal bacteria.More particularly, such ema mutant is a Group B streptococcal bacteria.Such non-adherent and/or non-invasive ema mutant bacteria can further beutilized in expressing other immunogenic or therapeutic proteins for thepurposes of eliciting immune responses to any such other proteins in thecontext of vaccines and in other forms of therapy.

Other objects and advantages will become apparent to those skilled inthe art from a review of the following description which proceeds withreference to the following illustrative drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the restriction digest pattern (RDP) type III-3 specificprobes. Dot blot hybridization of probe DY1-1 with genomic DNA isolatedfrom type III GBS. 10 ug of genomic DNA from each of 62 type III GBSstrains was transferred to nylon membrane. Radiolabeled probe DY1-1hybridized with DNA from all III-3 strains (rows A-D) including theoriginal type III-3 strain (well E-1). The probe failed to hybridizewith DNA from III-2 strains (F1-F10, G1-7) including the original strainused in the subtraction hybridization (well E 10) and III-1 strains(wells H1-3; cf. FIG. 3). The same pattern of hybridization was observedusing probe DY1-11.

FIG. 2 depicts the nucleic acid (SEQ ID NO:1) and predicted amino acid(SEQ ID NO:2) sequence of emaA.

FIGS. 3A and 3B depict the nucleic acid (SEQ ID NO:3) and predictedamino acid (SEQ ID NO:4) sequence of emaB.

FIG. 4 depicts the nucleic acid (SEQ ID NO:5) and predicted amino acid(SEQ ID NO:6) sequence of emaC.

FIGS. 5A and 5B depict the nucleic acid (SEQ ID NO:7) and predictedamino acid (SEQ ID NO:8) sequence of emaD.

FIGS. 6 A-D depict the nucleic acid (SEQ ID NO:9) and predicted aminoacid (SEQ ID NO:10) sequence of emaE.

DETAILED DESCRIPTION

The present invention provides novel Group B streptococcal Emapolypeptides and their Ema homologs in distinct bacterial species,including distinct streptococcal species. The present invention relatesto novel streptococcal Ema polypeptides, particularly selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE, and fragments thereof. Nucleicacids encoding Ema polypeptides, and diagnostic and therapeuticcompositions and methods based thereon for identification and preventionof infections by virulent forms of streptococci are provided. Inparticular, the present invention includes Group B streptococcal Emapolypeptides. The invention further includes polypeptide homologs of theGBS Ema polypeptides, particularly streptococcal homologs, moreparticularly Ema homologs of S. pneumoniae and S. pyogenes. BacterialEma polypeptide homologs in E. faecalis and C. diptheriae are alsoprovided.

Polypeptides

The present invention is directed to an isolated polypeptide comprisingan amino acid sequence of a bacterial Ema polypeptide. Bacterial Emapolypeptides are provided from streptococcus, enterococcus andcorynebacterium. The present invention is particularly directed to anisolated polypeptide comprising an amino acid sequence of astreptococcal Ema polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE. The present invention is particularly directed toan isolated polypeptide comprising an amino acid sequence of a Groupstreptococcal Ema polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE. Additional S. pneumoniae and S. pyogenes Emapolypeptides are included in the invention. E. faecalis and C.diptheriae Ema polypeptides are also included in the invention.

The polypeptides of the present invention are suitable for use inimmunizing animals broadly against streptococcal infection. Thepolypeptides of the present invention are suitable for use in immunizinganimals broadly against Group B, Group A, and S. pneumoniaestreptococcal infection. The polypeptides of the present invention aresuitable for use in immunizing animals against Group B streptococci.These polypeptide or peptide fragments thereof, when formulated with anappropriate adjuvant, are used in vaccines for protection againststreptococci, particularly Group B streptococci, and against otherbacteria with cross-reactive proteins.

GBS proteins with streptococcal homologs outside of Group B have beenpreviously identified (Lachenauer C S and Madoff L C (1997) Adv Exp Med.Biol. 418:615-8; Brady L. J. et al (1991) Infect Immun 59(12):4425-35;Stahlhammer-Carlemalm M. et al (2000) J Infect Dis 182(1):142-129).Stahlhammer-Carlemalm et al have demonstrated cross-protection betweenGroup A and Group B streptococci due to cross-reacting surface proteins(Stahlhammer-Carlemalm M. et al (2000) J Infect Dis 182(1):142-129). TheR28 protein of group A streptococcus (GAS) and the Rib protein of groupB streptococcus (GBS) are surface molecules that elicit protectiveimmunity to experimental infection. These proteins are members of thesame family and cross-react immunologically. In spite of extensive aminoacid residue identity, the cross-reactivity between R28 and Rib wasfound to be limited, as shown by analysis with highly purified proteinsand specific antisera. Nevertheless, immunization of mice with purifiedR28 conferred protection against lethal infection with Rib-expressingGBS strains, and immunization with Rib conferred protection againstR28-expressing GAS. Thus, R28 and Rib elicited cross-protectiveimmunity.

The present invention is directed to an isolated streptococcal EmaApolypeptide which comprises the amino acid sequence set out in SEQ IDNO: 2, and analogs, variants and immunogenic fragments thereof.

The present invention is directed to an isolated streptococcal EmaBpolypeptide which comprises the amino acid sequence set out in SEQ IDNO: 4, and analogs, variants and immunogenic fragments thereof.

The present invention is directed to an isolated streptococcal EmaCpolypeptide which comprises the amino acid sequence set out in SEQ IDNO: 6, and analogs, variants and immunogenic fragments thereof.

The present invention is directed to an isolated streptococcal EmaDpolypeptide which comprises the amino acid sequence set out in SEQ IDNO: 8, and analogs, variants and immunogenic fragments thereof.

The identity or location of one or more amino acid residues may bechanged or modified to include variants such as, for example, deletionscontaining less than all of the residues specified for the protein,substitutions wherein one or more residues specified are replaced byother residues and additions wherein one or more amino acid residues areadded to a terminal or medial portion of the polypeptide. Thesemolecules include: the incorporation of codons “preferred” forexpression by selected non-mammalian hosts; the provision of sites forcleavage by restriction endonuclease enzymes; and the provision ofadditional initial, terminal or intermediate DNA sequences thatfacilitate construction of readily expressed vectors.

The present invention is directed to an isolated Group B streptococcalEmaE polypeptide which comprises the amino acid sequence set out in SEQID NO: 10, and analogs, variants and immunogenic fragments thereof.

The present invention thus provides an isolated streptococcal Emapolypeptide comprising the amino acid sequence, set out in SEQ ID NO:23.An isolated nucleic acid which encodes the streptococcal polypeptide setout in SEQ ID NO:23 is further provided.

The invention thus further provides an isolated streptococcal Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO:26.An isolated nucleic acid which encodes the streptococcal polypeptide setout in SEQ ID NO:26 is further provided.

The present invention further provides an isolated streptococcal Emapolypeptide comprising the amino acid sequence set out in SEQ ID NO:37.An isolated nucleic acid which encodes the streptococcal polypeptide setout in SEQ ID NO:37 is further provided.

An enterococcal Ema polypeptide is further provided comprising the aminoacid sequence set out in SEQ ID NO:29. An isolated nucleic acid whichencodes the enterococcal polypeptide set out in SEQ ID NO:29 is alsoprovided.

The invention provides an isolated Corynebacterium Ema polypeptidecomprising the amino acid sequence set out in SEQ ID NO: 32. Alsoprovided is an isolated nucleic acid which encodes the Corynebacteriumpolypeptide set out in SEQ ID NO: 32.

The invention provides an isolated bacterial polypeptide comprising theamino acid sequence TLLTCTPYMINS/THRLLVR/KG (SEQ ID NO: 34), wherein thepolypeptide is not isolated from Actinomyces.

The invention further provides an isolated streptococcal polypeptidecomprising the amino acid sequence TLLTCTPYMINS/THRLLVR/KG (SEQ ID NO:34).

Also provided is an isolated bacterial polypeptide comprising the aminoacid sequence TLVTCTPYGINTHRLLVTA (SEQ ID NO: 35).

The present invention includes an isolated bacterial polypeptidecomprising the amino acid sequence TLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36).An isolated streptococcal polypeptide comprising the amino acid sequenceTLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36) is also provided.

The invention further includes an isolated polypeptide having the aminoacid sequence selected from the group of TLLTCTPYMNS/TH LLVRIKG (SEQ IDNO: 34), TLVTCTPYGINTHRLLVTA (SEQ ID NO: 35), and TLVTCTPYGVNTKRLLVRG(SEQ ID NO: 36).

The present invention contemplates the use of the streptococcalpolypeptides of the present invention in diagnostic tests and methodsfor determining and/or monitoring of streptococcal infection. Thus, thepresent invention provides an isolated GBS Ema polypeptide, particularlyselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, labeled witha detectable label.

In the instance where a radioactive label, such as the isotopes .sup.3H,sup.14C, .sup.32.P, 35S, .sup.36Cl, .sup 51Cr, .sup.57Co, .sup.5.Fe,.sup.90Y, .sup125I, .sup.131I, and .sup.186Re are used, known currentlyavailable counting procedures may be utilized. In the instance where thelabel is an enzyme, detection may be accomplished by any of thepresently utilized calorimetric, spectrophotometric,fluorospectro-photometric, amperometric or gasometric techniques knownin the art.

The present invention extends to an immunogenic bacterial Emapolypeptide. The present invention extends to an immunogenicstreptococcal Ema polypeptide, particularly selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE, or a fragment thereof. The presentinvention also extends to immunogenic GBS Ema polypeptides wherein suchpolypeptides comprise a combination of at least one immunogenic GBS Emapolypeptide, selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,or immunogenic polypeptide fragment thereof and GBS polypeptide Spb1,Spb2, C protein alpha antigen, Rib or immunogenic fragments thereof.

As defined herein, “adhesion” means noncovalent binding of a bacteria toa human cell or secretion that is stable enough to withstand washing.

The term “extracellular matrix adhesin”, “Ema”, “ema” and any variantsnot specifically listed, may be used herein interchangeably, and as usedthroughout the present application and claims refer to proteinaceousmaterial including single or multiple proteins, and extends to thoseproteins having the amino acid sequence data described herein andparticularly identified by (SEQ ID NOS: 2, 4, 6, 8, 10, 23, 26, 29, 32and 37), and the profile of activities set forth herein and in theClaims. In particular the Ema proteins provided herein include EmaA,EmaB, EmaC, EmaD and EmaE. The Ema proteins include bacterial Emahomologs. Bacterial Ema homologs include those from streptococcalspecies and other bacterial species. Accordingly, proteins andpolypeptides displaying substantially equivalent or altered activity arelikewise contemplated. These modifications may be deliberate, forexample, such as modifications obtained through site-directedmutagenesis, or may be accidental, such as those obtained throughmutations in hosts that are producers of one or more Ema polypeptide.Also, the term “extracellular matrix adhesin (Ema)” is intended toinclude within its scope proteins specifically recited herein as well asall substantially homologous analogs and allelic variations.

This invention provides an isolated immunogenic polypeptide comprisingan amino acid sequence of a bacterial Ema polypeptide. This inventionprovides an isolated immunogenic polypeptide comprising an amino acidsequence of a streptococcal Ema polypeptide, particularly selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE. It is contemplated by thisinvention that the immunogenic polypeptide has the amino acid sequenceset forth in any of SEQ ID NOS: 2, 4, 6, 8, 10, 23, 26, 29, 32 and 37,including immunogenic fragments, mutants, variants, analogs, orderivatives, thereof.

This invention is directed to analogs of the polypeptide which comprisethe amino acid sequence as set forth above. The analog polypeptide mayhave an N-terminal methionine or a polyhistidine optionally attached tothe N or COOH terminus of the polypeptide which comprise the amino acidsequence.

In another embodiment, this invention contemplates peptide fragments ofthe polypeptide which result from proteolytic digestion products of thepolypeptide. In another embodiment, the derivative of the polypeptidehas one or more chemical moieties attached thereto. In anotherembodiment the chemical moiety is a water soluble polymer. In anotherembodiment the chemical moiety is polyethylene glycol. In anotherembodiment the chemical moiety is mono-, di-, tri- or tetrapegylated. Inanother embodiment the chemical moiety is N-terminal monopegylated.

Attachment of polyethylene glycol (PEG) to compounds is particularlyuseful because PEG has very low toxicity in mammals (Carpenter et al.,1971). For example, a PEG adduct of adenosine deaminase was approved inthe United States for use in humans for the treatment of severe combinedimmunodeficiency syndrome. A second advantage afforded by theconjugation of PEG is that of effectively reducing the immunogenicty andantigenicity of heterologous compounds. For example, a PEG adduct of ahuman protein might be useful for the treatment of disease in othermammalian species without the risk of triggering a severe immuneresponse. The compound of the present invention may be delivered in amicroencapsulation device so as to reduce or prevent an host immuneresponse against the compound or against cells which may produce thecompound. The compound of the present invention may also be deliveredmicroencapsulated in a membrane, such as a liposome. Numerous activatedforms of PEG suitable for direct reaction with proteins have beendescribed. Useful PEG reagents for reaction with protein amino groupsinclude active esters of carboxylic acid or carbonate derivatives,particularly those in which the leaving groups are N-hydroxysuccinimide,p-nitrophenol, imidazole or 1-hydroxy-2-nitrobenzene-4-sulfonate. PEGderivatives containing maleimido or haloacetyl groups are usefulreagents for the modification of protein free sulfhydryl groups.Likewise, PEG reagents containing amino hydrazine or hydrazide groupsare useful. for reaction with aldehydes generated by periodate oxidationof carbohydrate groups in proteins.

In one embodiment, the amino acid residues of the polypeptide describedherein are preferred to be in the “L” isomeric form. In anotherembodiment, the residues in the “D” isomeric form can be substituted forany L-amino acid residue, as long as the desired functional property oflectin activity is retained by the polypeptide. NH.sub.2 refers to thefree amino group present at the amino terminus of a polypeptide. COOHrefers to the free carboxy group present at the carboxy terminus of apolypeptide. Abbreviations used herein are in keeping with standardpolypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969).

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues.

Synthetic polypeptide, prepared using the well known techniques of solidphase, liquid phase, or peptide condensation techniques, or anycombination thereof, can include natural and unnatural amino acids.Amino acids used for peptide synthesis may be standard Boc(N.sup.alpha.-amino protected N.sup.alpha.-t-butyloxycarbonyl) aminoacid resin with the standard deprotecting, neutralization, coupling andwash protocols of the original solid phase procedure of Merrifield(1963, J. Am. Chem. Soc. 85:2149-2154), or the base-labileN.sup.alpha.-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) aminoacids first described by Carpino and Han (1972, J. Org. Chem.37:3403-3409). Thus, polypeptide of the invention may comprise D-aminoacids, a combination of D- and L-amino acids, and various “designer”amino acids (e.g., .beta.-methyl amino acids, C.alpha.-methyl aminoacids, and N.alpha.-methyl amino acids, etc.) to convey specialproperties. Synthetic amino acids include ornithine for lysine,fluorophenylalanine for phenylalanine, and norleucine for leucine orisoleucine. Additionally, by assigning specific amino acids at specificcoupling steps, .alpha.-helices, .beta. turns, .beta. sheets,.gamma.-turns, and cyclic peptides can be generated.

In one aspect of the invention, the peptides may comprise a specialamino acid at the C-terminus which incorporates either a CO.sub.2H orCONH.sub.2 side chain to simulate a free glycine or a glycine-amidegroup. Another way to consider this special residue would be as a D or Lamino acid analog with a side chain consisting of the linker or bond tothe bead. In one embodiment, the pseudo-free C-terminal residue may beof the D or the L optical configuration; in another embodiment, aracemic mixture of D and L-isomers may be used.

In an additional embodiment, pyroglutamate may be included as theN-terminal residue of the peptide. Although pyroglutamate is notamenable to sequence by Edman degradation, by limiting substitution toonly 50% of the peptides on a given bead with N-terminal pyroglutamate,there will remain enough non-pyroglutamate peptide on the bead forsequencing. One of ordinary skill would readily recognize that thistechnique could be used for sequencing of any peptide that incorporatesa residue resistant to Edman degradation at the N-terminus. Othermethods to characterize individual peptides that demonstrate desiredactivity are described in detail infra. Specific activity of a peptidethat comprises a blocked N-terminal group, e.g., pyroglutamate, when theparticular N-terminal group is present in 50% of the peptides, wouldreadily be demonstrated by comparing activity of a completely (100%)blocked peptide with a non-blocked (0%) peptide.

In addition, the present invention envisions preparing peptides thathave more well defined structural properties, and the use ofpeptidomimetics, and peptidornimetic bonds, such as ester bonds, toprepare peptides with novel properties. In another embodiment, a peptidemay be generated that incorporates a reduced peptide bond, i.e.,R.sub.1-CH.sub.2-NH-R.sub.2, where R.sub.1 and R.sub.2 are amino acidresidues or sequences. A reduced peptide bond may be introduced as adipeptide subunit. Such a molecule would be resistant to peptide bondhydrolysis, e.g., protease activity. Such peptides would provide ligandswith unique function and activity, such as extended half-lives in vivodue to resistance to metabolic breakdown, or protease activity.Furthermore, it is well known that in certain systems constrainedpeptides show enhanced functional activity (Hruby, 1982, Life Sciences31:189-199; Hruby et al., 1990, Biochem J. 268:249-262); the presentinvention provides a method to produce a constrained peptide thatincorporates random sequences at all other positions.

A constrained, cyclic or rigidized peptide may be preparedsynthetically, provided that in at least two positions in the sequenceof the peptide an amino acid or amino acid analog is inserted thatprovides a chemical functional group capable of cross-linking toconstrain, cyclise or rigidize the peptide after treatment to form thecross-link. Cyclization will be favored when a turn-inducing amino acidis incorporated. Examples of amino acids capable of cross-linking apeptide are cysteine to form disulfide, aspartic acid to form a lactoneor a lactase, and a chelator such as .gamma.-carboxyl-glutamic acid(Gla) (Bachem) to chelate a transition metal and form a cross-link.Protected .gamma.-carboxyl glutamic acid may be prepared by modifyingthe synthesis described by Zee-Cheng and Olson (1980, Biophys. Biochem.Res. Commun. 94:1128-1132). A peptide in which the peptide sequencecomprises at least two amino acids capable of cross-linking may betreated, e.g., by oxidation of cysteine residues to form a disulfide oraddition of a metal ion to form a chelate, so as to cross-link thepeptide and form a constrained, cyclic or rigidized peptide.

The present invention provides strategies to systematically preparecross-links. For example, if four cysteine residues are incorporated inthe peptide sequence, different protecting groups may be used (Hiskey,1981, in The Peptides: Analysis, Synthesis, Biology, Vol. 3, Gross andMeienhofer, eds., Academic Press: New York, pp. 137-167; Ponsanti etal., 1990, Tetrahedron 46:8255-8266). The first pair of cysteine may bedeprotected and oxidized, then the second set may be deprotected andoxidized. In this way a defined set of disulfide cross-links may beformed. Alternatively, a pair of cysteine and a pair of collating aminoacid analogs may be incorporated so that the cross-links are of adifferent chemical nature.

The following non-classical amino acids may be incorporated in thepeptide in order to introduce particular conformational motifs:1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazmierski et al., 1991,J. Am. Chem. Soc. 113:2275-2283); (2S,35)-methyl-phenylalanine,(2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and(2R,3R)-methyl-phenylalanine (Kazmierski and Hruby, 1991, TetrahedronLett.); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis, 1989,Ph.D. Thesis, University of Arizona);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.,1989, J. Takeda Res. Labs. 43:53-76); .beta.-carboline (D and L)(Kazmierski, 1988, Ph.D. Thesis, University of Arizona); HIC (histidineisoquinoline carboxylic acid) (Zechel et al., 1991, Int. J. Pep. ProteinRes. 43); and HIC (histidine cyclic urea) (Dharanipragada).

The following amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a .beta.-turn inducingdipeptide analog (Kemp et al., 1985, J. Org. Chem. 50:5834-5838);.beta.-sheet inducing analogs (Kemp et al., 1988, Tetrahedron Lett.29:5081-5082); .beta.-turn inducing analogs (Kemp et al., 1988,Tetrahedron Lett. 29:5057-5060); .varies.-helix inducing analogs (Kempet al., 1988, Tetrahedron Lett. 29:4935-4938); .gamma.-turn inducinganalogs (Kemp et al., 1989, J. Org. Chem. 54:109:115); and analogsprovided by the following references: Nagai and Sato, 1985, TetrahedronLett. 26:647-650; DiMaio et al., 1989, J. Chem. Soc. Perkin Trans. p.1687; also a Gly-Ala turn analog (Kahn et al., 1989, Tetrahedron Lett.30:2317); amide bond isostere (Jones et al., 1988, Tetrahedron Lett.29:3853-3856); tretrazol (Zabrocki et al., 1988, J. Am. Chem. Soc.110:5875-5880); DTC (Samanen et al., 1990, Int. J. Protein Pep. Res.35:501:509); and analogs taught in Olson et al., 1990, J. Am. Chem. Sci.112:323-333 and Garvey et al., 1990, J. Org. Chem. 56:436.Conformationally restricted mimetics of beta turns and beta bulges, andpeptides containing them, are described in U.S. Pat. No. 5,440,013,issued Aug. 8, 1995 to Kahn.

The present invention further provides for modification orderivatization of the polypeptide or peptide of the invention.Modifications of peptides are well known to one of ordinary skill, andinclude phosphorylation, carboxymethylation, and acylation.Modifications may be effected by chemical or enzymatic means. In anotheraspect, glycosylated or fatty acylated peptide derivatives may beprepared. Preparation of glycosylated or fatty acylated peptides is wellknown in the art. Fatty acyl peptide derivatives may also be prepared.For example, and not by way of limitation, a free amino group(N-terminal or lysyl) may be acylated, e.g., myristoylated. In anotherembodiment an amino acid comprising an aliphatic side chain of thestructure—(CH.sub.2).sub.nCH.sub.3 may be incorporated in the peptide.This and other peptide-fatty acid conjugates suitable for use in thepresent invention are disclosed in U.K. Patent GB-8809162.4,International Patent Application PCT/AU89/00166, and reference 5, supra.

Chemical Moieties For Derivatization. Chemical moieties suitable forderivatization may be selected from among water soluble polymers. Thepolymer selected should be water soluble so that the component to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. Preferably, for therapeutic use of theend-product preparation, the polymer will be pharmaceuticallyacceptable. One skilled in the art will be able to select the desiredpolymer based on such considerations as whether the polymer/componentconjugate will be used therapeutically, and if so, the desired dosage,circulation time, resistance to proteolysis, and other considerations.For the present component or components, these may be ascertained usingthe assays provided herein.

The water soluble polymer may be selected from the group consisting of,for example, polyethylene glycol, copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols and polyvinyl alcohol. Polyethylene glycol propionaldenhyde mayhave advantages in manufacturing due to its stability in water.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 2 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The number of polymer molecules so attached may vary, and one skilled inthe art will be able to ascertain the effect on function. One maymono-derivative, or may provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to component or componentsmolecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted component or componentsand polymer) will be determined by factors such as the desired degree ofderivatization (e.g., mono, di-, tri-, etc.), the molecular weight ofthe polymer selected, whether the polymer is branched or unbranched, andthe reaction conditions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the component or components with consideration of effects onfunctional or antigenic domains of the protein. There are a number ofattachment methods available to those skilled in the art, e.g., EP 0 401384 herein incorporated by reference (coupling PEG to G-CSF), see alsoMalik et al., 1992, Exp. Hematol. 20:1028-1035 (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group include lysine residues andthe—terminal amino acid residues; those having a free carboxyl groupinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecule(s). Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

Nucleic Acids

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook et al, “Molecular Cloning:A Laboratory Manual” (1989); “Current Protocols in Molecular Biology”Volumes I-III [Ausubel, R. M., ed. (1994)]; “Cell Biology: A LaboratoryHandbook” Volumes I-III [J. E. Celis, ed. (1994))]; “Current Protocolsin Immunology” Volumes I-III [Coligan, J. E., ed. (1994)];“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “TranscriptionAnd Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “AnimalCell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells AndEnzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To MolecularCloning” (1984).

Mutations can be made in a nucleic acid encoding the polypeptide of thepresent invention such that a particular codon is changed to a codonwhich codes for a different amino acid. Such a mutation is generallymade by making the fewest nucleotide changes possible. A substitutionmutation of this sort can be made to change an amino acid in theresulting protein in a non-conservative manner (i.e., by changing thecodon from an amino acid belonging to a grouping of amino acids having aparticular size or characteristic to an amino acid belonging to anothergrouping) or in a conservative manner (i.e., by changing the codon froman amino acid belonging to a grouping of amino acids having a particularsize or characteristic to an amino acid belonging to the same grouping).Such a conservative change generally leads to less change in thestructure and function of the resulting protein. A non-conservativechange is more likely to alter the structure, activity or function ofthe resulting protein. The present invention should be considered toinclude sequences containing conservative changes which do notsignificantly alter the activity or binding characteristics of theresulting protein. Substitutes for an amino acid within the sequence maybe selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. Amino acids containing aromatic ring structures arephenylalanine, tryptophan, and tyrosine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Such alterations will not beexpected to affect apparent molecular weight as determined bypolyacrylamide gel electrophoresis, or isoelectric point.

Particularly preferred substitutions are:

Lys for Arg and vice versa such that a positive charge may bemaintained;

Glu for Asp and vice versa such that a negative charge may bemaintained;

Ser for Thr such that a free —OH can be maintained; and

Gln for Asn such that a free NH.sub.2 can be maintained.

Synthetic DNA sequences allow convenient construction of genes whichwill express analogs or “muteins”. A general method for site-specificincorporation of unnatural amino acids into proteins is described inNoren, et al. Science, 244:182-188 (April 1989). This method may be usedto create analogs with unnatural amino acids.

This invention provides an isolated nucleic acid encoding a polypeptidecomprising an amino acid sequence of a streptococcal Ema polypeptide.This invention provides an isolated nucleic acid encoding a polypeptidecomprising an amino acid sequence of a streptococcal Ema polypeptide.This invention provides an isolated nucleic acid encoding a polypeptidecomprising an amino acid sequence of a Group B streptococcal Emapolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.This invention provides an isolated nucleic acid encoding a polypeptidecomprising an amino acid sequence of a Group B streptococcal Ema proteinselected from the group of Ema proteins EmA, EmaB, EmaC. EmaD and EmaEas set forth in FIGS. 2-6. The invention provides an isolated nucleicacid encoding a polypeptide comprising an amino acid sequence of abacterial Ema polypeptide selected from the group of SEQ ID NO: 23, 26,29, 32 and 37. In particular embodiments the nucleic acid is set forthin any of SEQ ID NOS: 1, 3, 5, 7, 9, 24, 27, 30, and 33, includingfragments, mutants, variants, analogs, or derivatives, thereof. Thenucleic acid is DNA, cDNA, genomic DNA, RNA. Further, the isolatednucleic acid may be operatively linked to a promoter of RNAtranscription.

The present invention also relates to isolated nucleic acids, such asrecombinant DNA molecules or cloned genes, or degenerate variantsthereof, mutants, analogs, or fragments thereof, which encode theisolated polypeptide or which competitively inhibit the activity of thepolypeptide. The present invention further relates to isolated nucleicacids, such as recombinant DNA molecules or cloned genes, or degeneratevariants thereof, mutants, analogs, or fragments thereof, which encode aGBS Ema polypeptide, particularly selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE. Preferably, the isolated nucleic acid, whichincludes degenerates, variants, mutants, analogs, or fragments thereof,has a sequence as set forth in SEQ ID NOS: 1, 3, 5, 7 or 9. In a furtherembodiment of the invention, the DNA sequence of the recombinant DNAmolecule or cloned gene may be operatively linked to an expressioncontrol sequence which may be introduced into an appropriate host. Theinvention accordingly extends to unicellular hosts transformed with thecloned gene or recombinant DNA molecule comprising a DNA sequenceencoding an Ema protein, particularly selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE, and more particularly, the DNA sequences orfragments thereof determined from the sequences set forth above.

In a particular embodiment, the nucleic acid encoding the EmaApolypeptide has the sequence selected from the group comprising SEQ IDNO:1; a sequence that hybridizes to SEQ ID NO:1 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:1 or a sequence thathybridizes to SEQ ID NO:1 under moderate stringency hybridizationconditions; degenerate variants thereof, alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaA polypeptide has the sequence selected from thegroup comprising SEQ ID NO:1; a sequence complementary to SEQ ID NO:1;or a homologous sequence which is substantially similar to SEQ ID NO:1.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:1.

In a particular embodiment, the nucleic acid encoding the EmaBpolypeptide has the sequence selected from the group comprising SEQ IDNO:3; a sequence that hybridizes to SEQ ID NO:3 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:3 or a sequence thathybridizes to SEQ ID NO:3 under moderate stringency hybridizationconditions; degenerate variants thereof, alleles thereof, andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaB polypeptide has the sequence selected from thegroup comprising SEQ ID NO:3; a sequence complementary to SEQ ID NO:3;or a homologous sequence which is substantially similar to SEQ ID NO:3.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:3.

In a particular embodiment, the nucleic acid encoding the EmaCpolypeptide has the sequence selected from the group comprising SEQ IDNO:5; a sequence that hybridizes to SEQ ID NO:5 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:5 or a sequence thathybridizes to SEQ ID NO:5 under moderate stringency hybridizationconditions; degenerate variants thereof, alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaC polypeptide has the sequence selected from thegroup comprising SEQ ID NO:5; a sequence complementary to SEQ ID NO:5;or a homologous sequence which is substantially similar to SEQ ID NO:5.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:5.

In a particular embodiment, the nucleic acid encoding the EmaDpolypeptide has the sequence selected from the group comprising SEQ IDNO:7; a sequence that hybridizes to SEQ ID NO:7 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:7 or a sequence thathybridizes to SEQ ID NO:7 under moderate stringency hybridizationconditions; degenerate variants thereof, alleles thereof, andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaD polypeptide has the sequence selected from thegroup comprising SEQ ID NO:7; a sequence complementary to SEQ ID NO:7;or a homologous sequence which is substantially similar to SEQ ID NO:7.In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:7.

In a particular embodiment, the nucleic acid encoding the EmaEpolypeptide has the sequence selected from the group comprising SEQ IDNO:9; a sequence that hybridizes to SEQ ID NO:9 under moderatestringency hybridization conditions; DNA sequences capable of encodingthe amino acid sequence encoded by SEQ ID NO:9 or a sequence thathybridizes to SEQ ID NO:9 under moderate stringency hybridizationconditions; degenerate variants thereof; alleles thereof; andhybridizable fragments thereof. In a particular embodiment, the nucleicacid encoding the EmaE polypeptide has the sequence selected from thegroup comprising SEQ ID NO:9; a sequence complementary to SEQ ID NO:9;or a homologous sequence which is substantially similar to SEQ ID NO:9In a further embodiment, the nucleic acid has the sequence consisting ofSEQ ID NO:9.

A nucleic acid capable of encoding a GBS polypeptide EmaA, EmaB, EmaC,EmaD or EmaE which is a recombinant DNA molecule is further provided.Such a recombinant DNA molecule wherein the DNA molecule is operativelylinked to an expression control sequence is also provided herein.

The present invention relates to nucleic acid vaccines or DNA vaccinescomprising nucleic acids encoding immunogenic bacterial Emapolypeptides, particularly immunogenic streptococcal Ema polypeptides.The present invention relates to nucleic acid vaccines or DNA vaccinescomprising nucleic acids encoding immunogenic GBS Ema polypeptides,particularly selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.The present invention relates to nucleic acid vaccines or DNA vaccinescomprising nucleic acids encoding one or more immunogenic GBS Emapolypeptide or a fragment thereof or any combination of one or more Emapolypeptide EmaA, EmaB, EmaC, EmaD or EmaE with at least one other GBSpolypeptide, particularly wherein said other GBS polypeptide is selectedfrom the group of Spb1, Spb2, C protein alpha antigen, Rib andimmunogenic polypeptide fragments thereof.

The invention further relates to a vaccine for protection of an animalsubject from infection with a streptococcal bacterium comprising avector containing a gene encoding an Ema polypeptide, particularlyselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, operativelyassociated with a promoter capable of directing expression of the genein the subject. The invention further relates to a vaccine forprotection of an animal subject from infection with a Group Bstreptococcal bacterium comprising a vector containing a gene encodingan Ema polypeptide selected from the group of EmaA, EmaB, EmaC, EmaD andEmaE operatively associated with a promoter capable of directingexpression of the gene in the subject. The present invention furtherprovides a nucleic acid vaccine comprising a recombinant DNA moleculecapable of encoding a GBS polypeptide EmaA, EmaB, EmaC, EmaD or EmaE.

The present invention provides a vector which comprises the nucleic acidcapable of encoding a bacterial Ema polypeptide, particularly astreptococcal Ema polypeptide. The present invention provides a vectorwhich comprises the nucleic acid capable of encoding an Ema polypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE and apromoter. The present invention provides a vector which comprises thenucleic acid of any of SEQ ID NO: 1, 3, 5, 7, 9, 24, 27, 30, and 33, anda promoter. The invention contemplates a vector wherein the promotercomprises a bacterial, yeast, insect or mammalian promoter. Theinvention contemplates a vector wherein the vector is a plasmid, cosmid,yeast artificial chromosome (YAC), bacteriophage or eukaryotic viralDNA.

The present invention further provides a host vector system for theproduction of a polypeptide which comprises the vector capable ofencoding an Ema polypeptide, particularly selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE, in a suitable host cell. A host vectorsystem is provided wherein the suitable host cell comprises aprokaryotic or eukaryotic cell. A unicellular host transformed with arecombinant DNA molecule or vector capable of encoding an Emapolypeptide, particularly selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE, is thereby provided.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA” or “DNA molecule” refers to the polymeric form ofdeoxyribonucleotides (adenine, guanine, thyrnine, or cytosine) in itseither single stranded form, or a double-stranded helix. This termrefers only to the primary and secondary structure of the molecule, anddoes not limit it to any particular tertiary forms. Thus, this termincludes double-stranded DNA found, inter alia, in linear DNA molecules(e.g., restriction fragments), viruses, plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenontranscribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence in the case of eukaryoticmRNA.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, in theremainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5.times.SSC and65.degree. C. for both hybridization and wash. However, one skilled inthe art will appreciate that such “standard hybridization conditions”are dependent on particular conditions including the concentration ofsodium and magnesium in the buffer, nucleotide sequence length andconcentration, percent mismatch, percent formamide, and the like. Alsoimportant in the determination of “standard hybridization conditions” iswhether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA.Such standard hybridization conditions are easily determined by oneskilled in the art according to well known formulae, whereinhybridization is typically 10-20.degree. C. below the predicted ordetermined T.sub.m with washes of higher stringency, if desired.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding an Ema polypeptide EmaA, EmaB,EmaC, EmaD or EmaE which code for an Ema polypeptide having the sameamino acid sequence as any of SEQ ID NOS:2, 4, 6, 8 or 10, but which aredegenerate to any of SEQ ID NOS: 1, 3, 5, 7 or 9. By “degenerate to” ismeant that a different three-letter codon is used to specify aparticular amino acid. It is well known in the art that the followingcodons can be used interchangeably to code for each specific amino acid:TABLE-US-00002 Phenylalanine (Phe or F) UUU or UUC Leucine (Leu or L)UUA or UUG or CUU or CUC or CUA or CUG Isoleucine (Ile or I) AUU or AUCor AUA Methionine (Met or M) AUG Valine (Val or V) GUU or GUC of GUA orGUG Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC Proline(Pro or P) CCU or CCC or CCA or CCG Threonine (Thr or T) ACU or ACC orACA or ACG Alanine (Ala or A) GCU or GCG or GCA or GCG Tyrosine (Tyr orY) UAU or UAC Histidine (H is or H) CAU or CAC Glutamine (Gln or Q) CAAor CAG Asparagine (Asn or N) AAU or AAC Lysine (Lys or K) AAA or AAGAspartic Acid (Asp or D) GAU or GAC Glutamic Acid (Glu or E) GAA or GAGCysteine (Cys or C) UGU or UGC Arginine (Arg or R) CGU or CGC or CGA orCGG or AGA or AGG Glycine (Gly or G) GGU or GGC or GGA or GGG Tryptophan(Trp or W) UGG Termination codon UAA (ochre) or UAG (amber) or UGA(opal)

It should be understood that the codons specified above are for RNAsequences. The corresponding codons for DNA have a T substituted for U.

Mutations can be made in SEQ ID NOS: 1, 3, 5, 7 or 9 such that aparticular codon is changed to a codon which codes for a different aminoacid. Such a mutation is generally made by making the fewest nucleotidechanges possible. A substitution mutation of this sort can be made tochange an amino acid in the resulting protein in a non-conservativemanner (i.e., by changing the codon from an amino acid belonging to agrouping of amino acids having a particular size or characteristic to anamino acid belonging to another grouping) or in a conservative manner(i.e., by changing the codon from an amino acid belonging to a groupingof amino acids having a particular size or characteristic to an aminoacid belonging to the same grouping). Such a conservative changegenerally leads to less change in the structure and function of theresulting protein. A non-conservative change is more likely to alter thestructure, activity or function of the resulting protein. The presentinvention should be considered to include sequences containingconservative changes which do not significantly alter the activity orbinding characteristics of the resulting protein.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the genie.

Further this invention also provides a vector which comprises theabove-described nucleic acid molecule. The promoter may be, or isidentical to, a bacterial, yeast, insect or mammalian promoter. Further,the vector may be a plasmid, cosmid, yeast artificial chromosome (YAC),bacteriophage or eukaryotic viral DNA. Other numerous vector backbonesknown in the art as useful for expressing protein may be employed. Suchvectors include, but are not limited to: adenovirus, simian virus 40(SV40), cytomegalovirus (CMV), mouse mammary tumor virus (MMTV), Moloneymurine leukemia virus, DNA delivery systems, i.e. liposomes, andexpression plasmid delivery systems. Such vectors may be obtainedcommercially or assembled from the sequences described by methodswell-known in the art.

This invention also provides a host vector system for the production ofa polypeptide which comprises the vector of a suitable host cell. A widevariety of unicellular host cells are also useful in expressing the DNAsequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudonioiias, Bacillis, Streptomyces, fungi such as yeasts, and animalcells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidneycells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g.,Sf9), and human cells and plant cells in tissue culture.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAs, e.g., the numerous derivatives of phage .lamda., M13 andfilamentous single stranded phage DNA; yeast plasmids such as the 2 .mu.plasmid or derivatives thereof, vectors useful in eukaryotic cells, suchas vectors useful in insect or mammalian cells; vectors derived fromcombinations of plasmids and phage DNAs, such as plasmids that have beenmodified to employ phage DNA or other expression control sequences; andthe like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage .lamda., the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinaseor other glycolytic enzymes, the promoters of acid phosphatase (e.g.,Pho5), the promoters of the yeast .alpha.-mating factors, and othersequences known to control the expression of genes of prokaryotic oreukaryotic cells or their viruses, and various combinations thereof.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

This invention further provides a method of producing a polypeptidewhich comprises growing the above-described host vector system undersuitable conditions permitting the production of the polypeptide andrecovering the polypeptide so produced.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or“.mu.g” mean microgram, “mg” means milligram, “ul” or “.mu.l” meanmicroliter, “ml” means milliliter, “l” means liter.

The present invention extends to the preparation of antisenseoligonucleotides and ribozymes that may be used to interfere with theexpression of one or more Ema protein at the translational level. Thisapproach utilizes antisense nucleic acid and ribozymes to blocktranslation of a specific mRNA, either by masking that mRNA with anantisense nucleic acid or cleaving it with a ribozyme.

Antisense nucleic acids are DNA or RNA molecules that are complementaryto at least a portion of a specific mRNA molecule. (See Weintraub, 1990;Marcus-Sekura, 1988.) In the cell, they hybridize to that mRNA, forminga double stranded molecule. The cell does not translate an mRNA in thisdouble-stranded form. Therefore, antisense nucleic acids interfere withthe expression of mRNA into protein. Oligomers of about fifteennucleotides and molecules that hybridize to the AUG initiation codonwill be particularly efficient, since they are easy to synthesize andare likely to pose fewer problems than larger molecules when introducingthem into Ema-producing cells. Antisense methods have been used toinhibit the expression of many genes in vitro (Marcus-Sekura, 1988;Hambor et al., 1988).

Ribozymes are RNA molecules possessing the ability to specificallycleave other single stranded RNA molecules in a manner somewhatanalogous to DNA restriction endonucleases. Ribozymes were discoveredfrom the observation that certain mRNAs have the ability to excise theirown introns. By modifying the nucleotide sequence of these RNAs,researchers have been able to engineer molecules that recognize specificnucleotide sequences in an RNA molecule and cleave it (Cech, 1988).Because they are sequence-specific, only mRNAs with particular sequencesare inactivated.

Investigators have identified two types of ribozymes, Tetrahymena-typeand “hammerhead”-type. (Hasselhoff and Gerlach, 1988) Tetrahymena-typeribozymes recognize four-base sequences, while “hammerhead”-typerecognize eleven- to eighteen-base sequences. The longer the recognitionsequence, the more likely it is to occur exclusively in the target mRNAspecies. Therefore, hammerhead-type ribozymes are preferable toTetrahymena-type ribozymes for inactivating a specific mRNA species, andeighteen base recognition sequences are preferable to shorterrecognition sequences.

Antibodies

This invention further provides an antibody capable of specificallyrecognizing or binding to the isolated Ema polypeptide of the presentinvention. The antibody may be a monoclonal or polyclonal antibody.Further, the antibody may be labeled with a detectable marker that iseither a radioactive, calorimetric, fluorescent, or a luminescentmarker. The labeled antibody may be a polyclonal or monoclonal antibody.In one embodiment, the labeled antibody is a purified labeled antibody.Methods of labeling antibodies are well known in the art.

In a further aspect, the present invention provides a purified antibodyto a bacterial Ema polypeptide, particularly a streptococcal Emapolypeptide. In a still further aspect, the present invention provides apurified antibody to a Group B sreptococcal polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE.

Antibodies against the isolated polypeptides of the present inventioninclude naturally raised and recombinantly prepared antibodies. Thesemay include both polyclonal and monoclonal antibodies prepared by knowngenetic techniques, as well as bi-specific (chimeric) antibodies, andantibodies including other functionalities suiting them for diagnosticuse. Such antibodies can be used in immunoassays to diagnose infectionwith a particular strain or species of bacteria. The antibodies can alsobe used for passive immunization to treat an infection with Group Bstreptococcal bacteria. These antibodies may also be suitable formodulating bacterial adherence and/or invasion including but not limitedto acting as competitive agents.

The present invention provides a monoclonal antibody to a Group Bstreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE. The invention thereby extends to an immortal cell linethat produces a monoclonal antibody to a Group B streptococcalpolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.

An antibody to an Ema polypeptide, particularly selected from EmaA,EmaB, EmaC. EmaD or EmaE, labeled with a detectable label is furtherprovided. In particular embodiments, the label may selected from thegroup consisting of an enzyme, a chemical which fluoresces, and aradioactive element.

The term “antibody” includes, by way of example, both naturallyoccurring and non-naturally occurring antibodies. Specifically, the term“antibody” includes polyclonal and monoclonal antibodies, and fragmentsthereof. Furthermore, the term “antibody” includes chimeric antibodiesand wholly synthetic antibodies, and fragments thereof. Such antibodiesinclude but are not limited to polyclonal, monoclonal, chimeric, singlechain, Fab fragments, and an Fab expression library. An “antibody” isany immunoglobulin, including antibodies and fragments thereof, thatbinds a specific epitope. The term encompasses polyclonal, monoclonal,and chimeric antibodies, the last mentioned described in further detailin U.S. Pat. Nos. 4,816,397 and 4,816,567.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

The phrase “antibody molecule” in its various grammatical forms as usedherein contemplates both an intact immunoglobulin molecule and animmunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules,substantially intact immunoglobulin molecules and those portions of animmunoglobulin molecule that contains the paratope, including thoseportions known in the art as Fab, Fab′, F(ab′).sub.2 and F(v), whichportions are preferred for use in the therapeutic methods describedherein. Fab and F(ab′).sub.2 portions of antibody molecules are preparedby the proteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibody molecules by methods that are well-known.See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′antibody molecule portions are also well-known and are produced fromF(ab′).sub.2 portions followed by reduction of the disulfide bondslinking the two heavy chain portions as with mercaptoethanol, andfollowed by alkylation of the resulting protein mercaptan with a reagentsuch as iodoacetamide. An antibody containing intact antibody moleculesis preferred herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto an antibody having only one species of antibody combining sitecapable of immunoreacting with a particular antigen. A monoclonalantibody thus typically displays a single binding affinity for anyantigen with which it immunoreacts. A monoclonal antibody may thereforecontain an antibody molecule having a plurality of antibody combiningsites, each immunospecific for a different antigen; e.g., a bispecific(chimeric) monoclonal antibody.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to polypeptide or derivatives or analogs thereof(see, e.g., Antibodies—A Laboratory Manual, Harlow and Lane, eds., ColdSpring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1988). For theproduction of antibody, various host animals can be immunized byinjection with the Group B streptococcal Ema polypeptide, an immunogenicfragment thereof, or a derivative (e.g., fragment or fusion protein)thereof, including but not limited to rabbits, mice, rats, sheep, goats,etc. In one embodiment, the polypeptide can be conjugated to animmunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH). Various adjuvant may be used to increase theimmunological response, depending on the host species.

For preparation of monoclonal antibodies, or fragment, analog, orderivative thereof, any technique that provides for the production ofantibody molecules by continuous cell lines in culture may be used (see,e.g., Antibodies—A Laboratory Manual, Harlow and Lane, eds., Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y., 1988). These includebut are not limited to the hybridoma technique originally developed byKohler and Milstein (1975, Nature 256:495-497), as well as the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72), and the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96). Monoclonal antibodiescan be produced in germ-free animals utilizing recent technology(PCT/US90/02545). Human antibodies may be used and can be obtained byusing human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80:2026-2030) or by transforming human B cells with EBV virus in vitro(Cole et al., 1985, in Monoclonal Antitibodies and Cancer Therapy, AlanR. Liss, pp. 77-96). In fact, according to the invention, techniquesdeveloped for the production of “chimeric antibodies” (Morrison et al,1984, J. Bacteriol. 159-870; Neuberger et al, 1984, Nature 312:604-608;Takeda et al., 1985, Nature 314:452-454) by splicing the genes from amouse antibody molecule specific for a polypeptide together with genesfrom a human antibody molecule of appropriate biological activity can beused; such antibodies are within the scope of this invention. Such humanor humanized chimeric antibodies are preferred for use in therapy ofhuman infections or diseases, since the human or humanized antibodiesare much less likely than xenogenic antibodies to induce an immuneresponse, in particular an allergic response, themselves. An additionalembodiment of the invention utilizes the techniques described for theconstruction of Fab expression libraries (Huse et al., 1989, Science246:1275-1281) to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity for the polypeptide, or itsderivatives, or analogs.

Antibody fragments which contain the idiotype of the antibody moleculecan be generated by known techniques. For example, such fragmentsinclude but are not limited to: the F(ab′).sub.2 fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments which can be generated by reducing the disulfide bridges ofthe F(ab′).sub.2 fragment, and the Fab fragments which can be generatedby treating the antibody molecule with papain and a reducing agent.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., radioimmunoassay,ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays,immunoradiometric assays, gel diffusion precipitin reactions,immunodiffusion assays, in situ immunoassays (using colloidal gold,enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

Antibodies can be labeled for detection in vitro, e.g., with labels suchas enzymes, fluorophores, chromophores, radioisotopes, dyes, colloidalgold, latex particles, and chemiluminescent agents. Alternatively, theantibodies can be labeled for detection in vivo, e.g., withradioisotopes (preferably technetium or iodine); magnetic resonanceshift reagents (such as gadolinium and manganese); or radio-opaquereagents.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others. A number of fluorescent materials are known and canbe utilized as labels. These include, for example, fluorescein,rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. Aparticular detecting material is anti-rabbit antibody prepared in goatsand conjugated with fluorescein through an isothiocyanate. Thepolypeptide can also be labeled with a radioactive element or with anenzyme. The radioactive label can be detected by any of the currentlyavailable counting procedures. The preferred isotope may be selectedfrom .sup.3H, sup.14C, .sup.32P, .sup.35S, .sup.36Cl, sup 51CR,.sup.58Co, .sup.59Fe, .sup.90Y, .sup.125I, .sup131I, and .sup.186Re.

Enzyme labels are likewise useful, and can be detected by any of thepresently utilized calorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides, diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be tilized. The preferred are peroxidase, .beta.-glucuronidase,.beta.-D-glucosidase, .beta.-D-galactosidase, urease, glucose oxidaseplus peroxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090;3,850,752; and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

Diagnostic Applications

The present invention also relates to, a variety of diagnosticapplications, including methods for identifying or monitoringstreptococcal infections. The present invention also relates to avariety of diagnostic applications, including methods for identifying ormonitoring Group B streptococcal infections. The present inventionfurther relates to diagnostic applications or methods utilizing thepolypeptides of the present invention, immunogenically recognizedfragments thereof, or antibodies thereto. Such methods include theanalysis and evaluation of agents, analogs or compounds which modulatethe activity of the Ema polypeptides. The Ema polypeptides may also beutilized in diagnostic methods and assays for monitoring and determiningimmunological response and antibody response upon streptococcalinfection or vaccination.

As described in detail above, antibody(ies) to the Ema polypeptides orfragments thereof can be produced and isolated by standard methodsincluding the well known hybridoma techniques. For convenience, theantibody(ies) to the Ema polypeptides will be referred to herein asAb.sub.1, and antibody(ies) raised in another species as Ab.sub.2.

The presence of streptococci in cells can be ascertained by the usualimmunological procedures applicable to such determinations. A number ofuseful procedures are known. Procedures which are especially usefulutilize either the Ema polypeptides labeled with a detectable label,antibody against the Ema polypeptides labeled with a detectable label,or secondary antibody labeled with a detectable label.

The procedures and their application are all familiar to those skilledin the art and accordingly may be utilized within the scope of thepresent invention. The “competitive” procedure, is described in U.S.Pat. Nos. 3,654,090 and 3,850,752. The “sandwich” procedure, isdescribed in U.S. Pat. Nos. RE 31,006 and 4,016,043. Still otherprocedures are known such as the “double antibody,” or “DASP” procedure.

In each instance, the Ema polypeptides forms complexes with one or moreantibody(ies) or binding partners and one member of the complex islabeled with a detectable label. The fact that a complex has formed and,if desired, the amount thereof, can be determined by known methodsapplicable to the detection of labels.

In a further embodiment of this invention, commercial test kits suitablefor use by a medical specialist may be prepared to determine thepresence or absence of streptococci, particularly of streptococciexpressing one or more Ema polypeptide selected from the group of EmaA,EmaB, EmaC, EmaD and EmaE. In as much as the ema locus, as describedherein, is found in the genomic DNA of many, if not all, serotypes ofGroup B streptococci, it is a useful general marker for Group Bstreptococci. In as much as Ema homologs exist in other species ofstreptococci, including Group A and S. pneumoniae, it is a usefulgeneral marker for streptococci. Therefore, commercial test kits fordetermining the presence or absence of streptococci, and therebydetermining whether an individual is infected with streptococci arecontemplated and provided by this invention. Therefore, commercial testkits for determining the presence or absence of Group B streptococci,and thereby determining whether an individual is infected with Group Bstreptococci are contemplated and provided by this invention.

The present invention includes methods for determining and monitoringinfection by streptococci by detecting the presence of a streptococcalpolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE.In a particular such method, the streptococcal Ema polypeptide ismeasured by:

a. contacting a sample in which the presence or activity of aStreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE is suspected with an antibody to the said streptococcalpolypeptide under conditions that allow binding of the streptococcalpolypeptide to the antibody to occur; and

b. detecting whether binding has occurred between the streptococcalpolypeptide from the sample and the antibody; wherein the detection ofbinding indicates the presence or activity of the streptococcalpolypeptide in the sample.

The present invention includes methods for determining and monitoringinfection by 10 Group B streptococci by detecting the presence of aGroup B streptococcal polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE. In a particular such method, the streptococcal Emapolypeptide is measured by:

a. contacting a sample in which the presence or activity of a Group BStreptococcal polypeptide selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE is suspected with an antibody to the said Group Bstreptococcal polypeptide under conditions that allow binding of theGroup B streptococcal polypeptide to the antibody to occur; and

b. detecting whether binding has occurred between the Group Bstreptococcal polypeptide from the sample and the antibody; wherein thedetection of binding indicates the presence or activity of the a Group Bstreptococcal polypeptide in the sample.

The present invention further provides a method for detecting thepresence of a bacterium having a gene encoding a Group B polypeptideselected from the group of emaA, emaB, emaC, emaD and emaE, comprising:

a. contacting a sample in which the presence or activity of thebacterium is suspected with an oligonucleotide which hybridizes to aGroup B streptococcal polypeptide gene selected from the group of emaA,emaB, emaC, emaD and emaE, under conditions that allow specifichybridization of the oligonucleotide to the gene to occur; and

b. detecting whether hybridization has occurred between theoligonucleotide and the gene; wherein the detection of hybridizationindicates that presence or activity of the bacterium in the sample.

The invention includes an assay system for screening of potentialcompounds effective to modulate the activity of a bacterial Ema proteinof the present invention. In one instance, the test compound, or anextract containing the compound, could be administered to a cellularsample expressing the particular Ema protein to determine the compound'seffect upon the activity of the protein by comparison with a control. Ina further instance the test compound, or an extract containing thecompound, could be administered to a cellular sample expressing the Emaprotein to determine the compound's effect upon the activity of theprotein, and thereby on adherence of said cellular sample to host cells,by comparison with a control.

Accordingly, a test kit may be prepared for the demonstration of thepresence of Ema polypeptide or Ema activity in cells, comprising:

a. a predetermined amount of at least one labeled immunochemicallyreactive component obtained by the direct or indirect attachment of theEma polypeptide or a specific binding partner thereto, to a detectablelabel;

b. other reagents; and

c. directions for use of said kit.

More specifically, the diagnostic test kit may comprise:

a. a known amount of the Ema polypeptide as described above (or abinding partner) generally bound to a solid phase to form animmunosorbent, or in the alternative, bound to a suitable tag, or pluralsuch end products, etc. (or their binding partners) one of each;

b. if necessary, other reagents; and

c. directions for use of said test kit.

In a further variation, the test kit may be prepared and used for thepurposes stated above, which operates according to a predeterminedprotocol (e.g. “competitive,” “sandwich,” “double antibody,” etc.), andcomprises:

a. a labeled component which has been obtained by coupling the Emapolypeptide to a detectable label;

b. one or more additional immunochemical reagents of which at least onereagent is a ligand or an immobilized ligand, which ligand is selectedfrom the group consisting of:

-   -   (i) a ligand capable of binding with the labeled component a.;    -   (ii) a ligand capable of binding with a binding partner of the        labeled component a.;    -   (iii) a ligand capable of binding with at least one of the        component(s) to be determined; and    -   (iv) a ligand capable of binding with at least one of the        binding partners of at least one of the component(s) to be        determined; and

c. directions for the performance of a protocol for the detection and/ordetermination of one or more components of an immunochemical reactionbetween the Ema polypeptide and a specific binding partner thereto.

In accordance with the above, an assay system for screening potentialdrugs effective to modulate the activity of the Ema polypeptide may beprepared. The Ema polypeptide may be introduced into a test system, andthe prospective drug may also be introduced into the resulting cellculture, and the culture thereafter examined to observe any changes inthe Ema polypeptide activity of the cells, due either to the addition ofthe prospective drug alone, or due to the effect of added quantities ofthe known Ema polypeptide.

Therapeutic Applications

The therapeutic possibilities that are raised by the existence of theGroup B streptococcal Ema polypeptides EmaA, EmaB, EmaC, EmaD and EmaEderive from the fact that the Ema polypeptides of the present inventionare found generally in various serotypes of Group B streptococci. Inaddition, broader therapeutic possibilities that are raised by theexistence of Ema homologous polypeptides in various distinct species ofstreptococci including S. pneumoniae and S. pyogenes. In addition Emahomologous polypeptides have been identified in E. faecalis and C.diptheriae. Of particular relevance to their suitability in vaccine andimmunological therapy is that the Ema A, EmaB, and EmaC polypeptidespossess N-terminal sequences consistent with a signal peptide,indicating secretion from the bacterial cell and at least partialextracellular localization. In addition, the EmaA, EmaB, EmaC, EmaD andEmaE polypeptides demonstrate homology to distinct bacterial proteinsinvolved in or implicated in bacterial adhesion and invasion. Thus, theEma polypeptides are anticipated to be involved in or required forstreptococcal adhesion to and/or invasion of cells, critical forbacterial survival and virulence in the human host.

Modulators of Extracellular Matrix Adhesin Protein

Thus, in instances where it is desired to reduce or inhibit the effectsresulting from the extracellular matrix adhesin protein Ema of thepresent invention, an appropriate inhibitor of one or more of the Emaproteins, particularly EmaA, EmaB, EmaC, EmaD and EmaE could beintroduced to block the activity of one or more Ema protein.

The present invention contemplates screens for a modulator of an Emapolypeptide, in particular modulating adhesion or invasion facilitatedby EmaA, EmaB, EmaC, EmaD or EmaE. In one such embodiment, an expressionvector containing the Ema polypeptide of the present invention, or aderivative or analog thereof, is placed into a cell in the presence ofat least one agent suspected of exhibiting Ema polypeptide modulatoractivity. The cell is preferably a bacterial cell, most preferably astreptococcal cell, or a bacterial host cell. The amount of adhesion orbinding activity is determined and any such agent is identified as amodulator when the amount of adhesion or binding activity in thepresence of such agent is different than in its absence. The vectors maybe introduced by any of the methods described above. In a relatedembodiment the GBS Ema polypeptide is expressed in streptococci and thestep of determining the amount of adhesion or binding activity isperformed by determining the amount of binding to bacterial host cellsin vitro.

When the amount of adhesion or binding activity in the presence of themodulator is greater than in its absence, the modulator is identified asan agonist or activator of the Ema polypeptide, whereas when the amountof adhesion binding activity in the presence of the modulator is lessthan in its absence, the modulator is identified as an antagonist orinhibitor of the Ema polypeptide. As any person having skill in the artwould recognize, such determinations as these and those below couldrequire some form of statistical analysis, which is well within theskill in the art.

Natural effectors found in cells expressing Ema polypeptide can befractionated and tested using standard effector assays as exemplifiedherein, for example. Thus an agent that is identified can be a naturallyoccurring adhesion or binding modulator. Alternatively, natural productslibraries can be screened using the assays of the present invention forscreening such agents.

Another approach uses recombinant bacteriophage to produce largelibraries. Using the “phage method” [Scott and Smith, 1990, Science249:386-390 (1990); Cwirla, et al., Proc. Natl. Acad. Sci., 87:6378-6382(1990); Devlin et al., Science, 249:404-406 (1990)], very largelibraries can be constructed (10.sup.6-10.sup.8 chemical entities). Yetanother approach uses primarily chemical methods, of which the Geysenmethod [Geysen et al., Molecular Immunology 23:709-715 (1986); Geysen etal. J. Immunologic Method 102:259-274 (1987)] and the method of Fodor etal. [Science 251:767-773 (1991)] are examples. Furka et al. [14thInternational Congress of Biochemistry, Volume 5, Abstract FR:013(1988); Furka, Int. J. Peptide Protein Res. 37:487-493 (1991)], Houghton[U.S. Pat. No. 4,631,211, issued December 1986] and Rutter et al. [U.S.Pat. No. 5,010,175, issued Apr. 23, 1991] describe methods to produce amixture of peptides that can be tested.

In another aspect, synthetic libraries [Needels et al., Proc. Natl.Acad. Sci. USA 90:10700-4 (1993); Ohlmeyer et al., Proc. Natl. Acad.Sci. USA 90:10922-10926 (1993); Lam et al., International PatentPublication No. WO 92/00252; Kocis et al., International PatentPublication No. WO 9428028, each of which is incorporated herein byreference in its entirety], and the like can be used to screen for suchan agent.

This invention provides antagonist or blocking agents which include butare not limited to: peptide fragments, mimetic, a nucleic acid molecule,a ribozyme, a polypeptide, a small molecule, a carbohydrate molecule, amonosaccharide, an oligosaccharide or an antibody. Also, agents whichcompetitively block or inhibit streptococcal bacterium are contemplatedby this invention. This invention provides an agent which comprises aninorganic compound, a nucleic acid molecule, an oligonucleotide, anorganic compound, a peptide, a peptidomimetic compound, or a proteinwhich inhibits the polypeptide.

Vaccines

In a further aspect, the present invention extends to vaccines based onthe Ema proteins described herein. The present invention provides avaccine comprising one or more Group B streptococcal polypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, and apharmaceutically acceptable adjuvant. The present invention provides avaccine comprising one or more bacterial Ema polypeptide selected fromthe group of polypeptides comprising the amino acid sequence set out inany of SEQ ID NO: 23, 26, 29, 32 and 37, and a pharmaceuticallyacceptable adjuvant.

The present invention further provides a vaccine comprising one or moreGroup B streptococcal polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE, further comprising one or more additional GBSantigen. The present invention further provides a vaccine comprising oneor more Group B streptococcal polypeptide selected from the group ofEmaA, EmaB, EmaC, EmaD and EmaE, further comprising one or more antigensselected from the group of the polypeptide Spb1 or an immunogenicfragment thereof, the polypeptide Spb2 or an immunogenic fragmentthereof, C protein alpha antigen or an immunogenic fragment thereof, Ribor an immunogenic fragment thereof, Lmb or an immunogenic fragmentthereof, C5a-ase or an immunogenic fragment thereof, and Group Bstreptococcal polysaccharides or oligosaccharides.

In another aspect, the invention is directed to a vaccine for protectionof an animal subject from infection with streptococci comprising animmunogenic amount of one or more streptococcal Ema polypeptide, or aderivative or fragment thereof. The Ema polypeptide may be particularlyselected from the group of EmaA, EmaB, EmaC, EmaD or EmaE, or aderivative or fragment thereof. In a further aspect, the invention isdirected to a vaccine for protection of an animal subject from infectionwith streptococci comprising an immunogenic amount of one or more Emapolypeptide EmaA, EmaB, EmaC, EmaD or EmaE, or a derivative or fragmentthereof. In a further aspect, the invention is directed to a vaccine forprotection of an animal subject from infection with GBS comprising animmunogenic amount of one or more Ema polypeptide EmaA, EmaB, EmaC, EmaDor EmaE, or a derivative or fragment thereof. Such a vaccine may containthe protein conjugated covalently to a streptococcal or GBS bacterialpolysaccharide or oligosaccharide or polysaccharide or oligosaccharidefrom one or more streptococcal or GBS serotypes.

This invention provides a vaccine which comprises a polypeptidebacterial Ema protein and a pharmaceutically acceptable adjuvant orcarrier. In particular, a vaccine is provided which comprises one ormore Ema polypeptides selected from the group of EmaA, EmaB, EmaC, EmaDand EmaE. This invention provides a vaccine which comprises acombination of at least one bacterial Ema protein selected from thegroup of EmaA, EmaB, EmaC, EmaD and EmaE and at least one other Group Bstreptococcal protein particularly Spb1 and/or Spb2 and/or C proteinalpha antigen, and a pharmaceutically acceptable adjuvant or carrier.The Ema polypeptide may comprise an amino acid sequence of a Ema proteinEmaA, EmaB, EmaC, EmaD, EmaE as set forth in FIGS. 2-6 and SEQ ID NOS:2, 4, 6, 8 and 10.

This invention further provides a vaccine comprising an isolated nucleicacid encoding a bacterial Ema polypeptide and a pharmaceuticallyacceptable adjuvant or carrier. This invention further provides avaccine comprising an isolated nucleic acid encoding a streptococcal Emapolypeptide and a pharmaceutically acceptable adjuvant or carrier. Thisinvention further provides a vaccine comprising an isolated nucleic acidencoding a GBS Ema polypeptide and a pharmaceutically acceptableadjuvant or carrier. This invention further provides a vaccinecomprising isolated nucleic acid encoding one or more GBS Emapolypeptide, particularly selected from the group of EmaA, EmaB, EmaC,EmaD and EmaE and a pharmaceutically acceptable adjuvant or carrier. Thenucleic acid may comprise a nucleic acid sequence of a GBS Emapolypeptide as set forth in any of SEQ ID NOS:1, 3, 5, 7, or 9.

Active immunity against streptococci can be induced by immunization(vaccination) with an immunogenic amount of the polypeptide, or peptidederivative or fragment thereof, and an adjuvant, wherein thepolypeptide, or antigenic derivative or fragment thereof, is theantigenic component of the vaccine. The polypeptide, or antigenicderivative or fragment thereof, may be one antigenic component, in thepresence of other antigenic components in a vaccine. For instance, thepolypeptide of the present invention may be combined with other knownstreptococcal polypeptides or poly/oligosaccharides, or immunogenicfragments thereof, including for instance GBS capsular polysaccharide,Spb1, Spb2, C protein alpha antigen, Rib, Lmb, and C5a-ase in amulti-component vaccine. Such multi-component vaccine may be utilized toenhance immune response, even in cases where the polypeptide of thepresent invention elicits a response on its own. The polypeptide of thepresent invention may also be combined with existing vaccines, wholebacterial or capsule-based vaccines, alone or in combination with otherGBS polypeptides, particularly Spb1 and/or Spb2 and/or C protein alphaantigen and/or Rib to enhance such existing vaccines.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response to an antigen. An adjuvant can serve as a tissue depotthat slowly releases the antigen and also as a lymphoid system activatorthat non-specifically enhances the immune response (Hood et al.,Immunology, Secoid Ed, 1984, Benjamin/Cummings: Menlo Park, Calif., p.384). Often, a primary challenge with an antigen alone, in 25 theabsence of an adjuvant, will fail to elicit a humoral or cellular immuneresponse.

Adjuvant include, but are not limited to, complete Freund's adjuvant,incomplete Freund's adjuvant, saponin, mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil or hydrocarbon emulsions, keyholelimpet hemocyanins, dinitrophenol, and potentially useful human adjuvantsuch as BCG (bacille Calniette-Guerin) and Corynebacterium parvum.Preferably, the adjuvant is pharmaceutically acceptable.

The invention further provides a vaccine which comprises a non-adherent,non-virulent mutant, including but not limited to the ema.sup.-mutantsherein described and contemplated. Medaglini et al (Madaglini et al(1995) Proc Natl Acad Sci USA 92; 6868-6872) and Oggioni and Pozzi(Oggioni, M. R. and Pozzi, G. (1996) Gene 169:85-90) have previouslydescribed the use of Streptococcus gordonii, a commensal bacterium ofthe human oral cavity, as live vaccine delivery vehicles and forheterologous gene expression. Such ema.sup.-mutant can therefore beutilized as a vehicle for expression of immunogenic proteins for thepurposes of eliciting an immune response to such other proteins in thecontext of vaccines. Active immunity against Group B streptococci, canbe induced by immunization (vaccination) with an immunogenic amount ofthe ema.sup.-vehicle expressing an immunogenic protein. Alsocontemplated by the present invention is the use of any suchema.sup.-mutant in expressing a therapeutic protein in the host in thecontext of other forms of therapy.

The polypeptide of the present invention, or fragments thereof, can beprepared in an admixture with an adjuvant to prepare a vaccine.Preferably, the polypeptide or peptide derivative or fragment thereof,used as the antigenic component of the vaccine is an antigen common toall or many serotypes of GBS bacteria, or common to closely relatedspecies of bacteria, for instance Streptococcus.

Vectors containing the nucleic acid-based vaccine of the invention canbe introduced into the desired host by methods known in the art, e.g.,transfection, electroporation, micro injection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a DNA vector transporter (see,e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J.Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent ApplicationNo. 2,012,311, filed Mar. 15, 1990).

The modes of administration of the vaccine or compositions of thepresent invention may comprise the use of any suitable means and/ormethods for delivering the vaccine or composition to the host animalwhereby they are immumostimulatively effective. Delivery modes mayinclude, without limitation, parenteral administration methods, such asparacancerally, transmucosally, transdermally, intramuscularly,intravenously, intradermally, subcutaneously, intraperitonealy,intraventricularly, intracranially and intratumorally. Preferably, sincethe desired result of vaccination is to elucidate an immune response tothe antigen, and thereby to the pathogenic organism, administrationdirectly, or by targeting or choice of a viral vector, indirectly, tolymphoid tissues, e.g., lymph nodes or spleen, is desirable. Sinceimmune cells are continually replicating, they are ideal target forretroviral vector-based nucleic acid vaccines, since retrovirusesrequire replicating cells. These vaccines and compositions can be usedto immunize mammals, for example, by the intramuscular or parenteralroutes, or by delivery to mucosal surfaces using microparticles,capsules, liposomes and targeting molecules, such as toxins andantibodies. The vaccines and immunogenic compositions may beadministered to mucosal surfaces by, for example, the nasal or oral(intragastric) routes. Alternatively, other modes of administrationincluding suppositories may be desirable. For suppositories, binders andcarriers may include, for example, polyalkylene glycols andtriglycerides. Oral formulations may include normally employedincipients, such as pharmaceutical grades of saccharine, cellulose andmagnesium carbonate.

These compositions may take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders and contain 1to 95% of the immunogenic compositions of the present invention. Theimmunogenic compositions are administered in a manner compatible withthe dosage formulation, and in such amount as to be therapeuticallyeffective, protective and immunogenic. The quantity to be administereddepends on the subject to the immunized, including, for example, thecapacity of the subject's immune system to synthesize antibodies, and ifneeded, to produce a cell-mediated, humoral or antibody-mediated immuneresponse. Precise amounts of antigen and immunogenic composition to beadministered depend on the judgement of the practitioner. However,suitable dosage ranges are readily determinable by those skilled in theart and may be of the order of micrograms to milligrams. Suitableregimes for initial administration and booster doses are also variable,but may include an initial administration followed by subsequentadministrations. The dosage of the vaccine may also depend on the routeof administration and will vary according to the size of the host.

Passive immunity can be conferred to an animal subject suspected ofsuffering an infection with streptococci by administering antiserum,polyclonal antibodies, or a neutralizing monoclonal antibody against oneor more Ema polypeptide of the invention to the patient. A combinationof antibodies directed against one or more Ema polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, in combination with one ormore of antibodies against Spb1, Spb2, Rib and C protein alpha antigenis also contemplated by the present invention. Although passive immunitydoes not confer long term protection, it can be a valuable tool for thetreatment of a bacterial infection in a subject who has not beenvaccinated. Passive immunity is particularly important for the treatmentof antibiotic resistant strains of bacteria, since no other therapy maybe available. Preferably, the antibodies administered for passive immunetherapy are autologous antibodies. For example, if the subject is ahuman, preferably the antibodies are of human origin or have been“humanized,” in order to minimize the possibility of an immune responseagainst the antibodies. The active or passive vaccines of the inventioncan be used to protect an animal subject from infection bystreptococcus, particularly Group B streptococcus.

Vaccines for GBS have been previously generated and tested. Preliminaryvaccines used unconjugated purified polysaccaride. GBS polysaccharidesand oligosaccharides are poorly immunogenic and fail to elicitsignificant memory and booster responses. Baker et al immunized 40pregnant women with purified serotype III capsular polysaccharide(Baker, C. J. et al. (1998) New Engl J of Med 319:1180-1185). Overall,only 57% of women with low levels of specific antibody responded to thevaccine. The poor immunogenicity of purified polysaccharide antigen wasfurther demonstrated in a study in which thirty adult volunteers wereimmunized with a tetravalent vaccine composed of purified polysaccharidefrom serotypes Ia, Ib, II, and III (Kotloff, K. L. et al. (1996) Vaccine14:446-450). Although safe, this vaccine was only modestly immunogenic,with only 13% of subjects responding to type Ib, 17% to type II, 33%responding to type Ia, and 70% responding to type III polysaccharide.The poor immunogenicity of polysaccaride antigens prompted efforts todevelop polysaccharide conjugate vaccines, whereby these polysaccharidesor oligosaccharides are conjugated to protein carriers. Ninety percentof healthy adult women immunized with a type III polysaccharide-tetanustoxoid conjugate vaccine responded with a 4-fold rise in antibodyconcentration, compared to 50% immunized with plain polysaccharide(Kasper, D. L. et al (1996) J of Clin Invest 98:2308-2314). A type Ia/Ibpolysaccharide-tetanus toxoid conjugate vaccine was similarly moreimmunogenic in healthy adults than plain polysaccharide (Baker, C. J. etal (1999) J Infect Dis 179:142-150).

The general method for the conjugation of polysaccharide is described inWessels et al (Wessels, M. R. et al (1990) J. Clin Investigation 86:1428-1433). Prior to coupling with tetanus toxoid, aldehyde groups areintroduced on the polysaccharide by controlled periodate oxidation,resulting in the conversion of a portion of the sialic acid residues ofthe polysaccharide to residues of the 8-carbon analogue of sialic acid,5-acetamido-3,5-dideoxy-D-galactosyloctulosonic acid. Tetanus toxoid isconjugated to the polysaccharide by reductive amination using freealdehyde groups present on the partially oxidized sialic acid residues.The preparation and conjugation of oligosaccharides is described inPaoletti et al (Paoletti, L. C. et al (1990) J. Biol Chem 265:18278-18283). Purified capsular polysaccharide is depolymerized byenzymatic digestion using endo-beta-galactosidase produced byCitrobacter freundii. Following digestion, oligosaccharides arefractionated by gel filtration chromatography. Tetanus toxoid wascovalently coupled via a synthetic spacer molecule to the reducing endof the oligosaccharide by reductive amination.

Methods and vaccines comprising GBS conjugate vaccines, comprisingcapsular polysaccharide and protein are provided and described in U.S.Pat. Nos. 5,993,825, 5,843,461, 5,795,580, 5,302,386 and 4,356,263,which are incorporated herein by reference in their entirety. Theseconjugate vaccines include polysaccharide-tetanus toxoid conjugatevaccines.

One polypeptide proposed to be utilized in a GBS vaccine is therepetitive GBS C protein alpha antigen, which contains up to ninetandemly repeated units of 82 amino acids (Michel, J. K. et al (1992)PNAS USA 89: 10060-10064). The polypeptide, methods and vaccinesthereof, including polysaccharide-conjugate vaccines generatedtherewith, are provided and described in U.S. Pat. Nos. 5,968,521,5,908,629, 5,858,362, 5,847,081, 5,843,461, 5,843,444, 5,820,860, and5,648,241, which are herein incorporated by reference in their entirety.Antibodies generated against C protein alpha antigen with a largenumbers of repeats protect against infection, but GBS are able to changethe structure of the protein by deleting one or more of the repeatregions and escape detection by these antibodies (Madoff, L. C. et al(1996) PNAS USA 93: 4131-4136). This effect could theoretically beprevented by immunization with a protein with a lower number of repeatunits, but the immunogenicity of the C protein alpha antigen isinversely related to the number of repeats—65% of mice responded toimmunization with the 9-repeat protein, but only 11% to a 1-repeatprotein (Cravekamp, C. et al (1997) Infect Immunity 65: 5216-5221). Thisis a disadvantage with any protein with a repetitive structure—it iscommon for bacteria to be able to alter or reassort these genes to alterthe proteins exposed on their surface.

Typical doses for a vaccine composed of a protein antigen are in therange of 2.5-50 ug of total protein per dose. Typical doses for apolysaccharide-protein conjugate vaccine are 7.5-25 ug of polysaccharideand 1.25-250 ug of carrier protein. These types of vaccines are almostalways given intramuscularly. Dosing schedules of a vaccine can bereadily determined by the skilled artisan, particularly by comparison ofsimilar vaccines, including other GBS vaccines. If used as a universalvaccine, a GBS vaccine would be integrated into the routine immunizationschedule. Most similar vaccines require a primary series ofimmunizations (usually 2 or 3 doses at 2 month intervals beginning at 1or 2 months of age) and a single booster at 12-18 months of age. Asmaller number of doses or a single dose may be adequate in olderchildren (over a year of age). For immunization of pregnant women, anexemplary immunization schedule would be a single dose given in thesecond or early third trimester. For immunization of non-pregnantadults, a single dose would probably be used. The requirement forsubsequent booster doses in adults is difficult to predict—this would bebased on the immunogenicity of the vaccine and ongoing surveillance ofvaccine efficacy.

Immunogenic Compositions

In a further aspect, the present invention provides an immunogeniccomposition comprising one of more bacterial Ema polypeptides. In astill further aspect, the present invention provides an immunogeniccomposition comprising one of more streptococcal Ema polypeptides. In aparticular aspect, the present invention provides an immunogeniccomposition comprising one of more Group B streptococcal polypeptidesselected from the group of EmaA, EmaB, EmaC, EmaD, EmaE and a fragmentthereof, and a pharmaceutically acceptable adjuvant. Immunogeniccompositions may comprise a combination of one or more Group B Emapolypeptide, or an immunogenic polypeptide fragment thereof, with one ormore additional GBS polypeptide or GBS capsular polysaccharide oroligosaccharide.

The present invention further provides an immunogenic compositioncomprising one or more Group B streptococcal polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, further comprising one ormore antigens selected from the group of the polypeptide Spb1 or animmunogenic fragment thereof, the polypeptide Spb2 or an immunogenicfragment thereof, C protein alpha antigen or an immunogenic fragmentthereof, Rib or an immunogenic fragment thereof, and Group Bstreptococcal polysaccharides or oligosaccharides.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising abacterial Ema polypeptide, particularly a streptococcal Ema polypeptide,and a pharmaceutically acceptable carrier. The invention providespharmaceutical compositions comprising a Group B streptococcalpolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,and a pharmaceutically acceptable carrier. The present invention furtherprovides pharmaceutical compositions comprising one or more GBS Emapolypeptide, or a fragment thereof, in combination with one or more ofGBS polypeptide Spb1, Spb2, C protein alpha antigen, Rib, a Group Bstreptococcal polysaccharide or oligosaccharide vaccine, and ananti-streptococcal vaccine.

Such pharmaceutical composition for preventing streptococcal attachmentto mucosal surface may include antibody to Ema polypeptide EmaA, EmaB,EmaC, EmaD or EmaE or any combination of antibodies to one or more suchEma polypeptide. In addition, any such composition may further includeantibody to GBS polypeptides Spb1, Spb2, C protein alpha antigen, orRib. Blocking adherence using such antibody blocks the initial step ininfection thereby reducing colonization. This in turn decreases personto person transmission and prevents development of symptomatic disease.

The present invention provides a pharmaceutical composition comprisingan antibody to a Group B streptococcal protein selected from the groupof EmaA, EmaB, EmaC, EmaD and EmaE, and a pharmaceutically acceptablecarrier. The invention further provides a pharmaceutical compositioncomprising a combination of at least two antibodies to Group Bstreptococcal proteins and a pharmaceutically acceptable carrier,wherein at least one antibody to a protein selected from the group ofEmaA, EmaB, EmaC, EmaD, EmaE, is combined with at least one antibody toa protein selected from the group of Spb1, Spb2, Rib, and C proteinalpha antigen.

It is still a further object of the present invention to provide amethod for the prevention or treatment of mammals to control the amountor activity of streptococci, so as to treat or prevent the adverseconsequences of invasive, spontaneous, or idiopathic pathologicalstates.

It is still a further object of the present invention to provide amethod for the prevention or treatment of mammals to control the amountor activity of Group B streptococci, so as to treat or prevent theadverse consequences of invasive, spontaneous, or idiopathicpathological states.

The invention provides a method for preventing infection with abacterium that expresses a streptococcal Ema polypeptide comprisingadministering an immunogenically effective dose of a vaccine comprisingan Ema polypeptide selected from the group of EmaA, EmaB, EmaC, EmaD andEmaE to a subject.

The invention further provides a method for preventing infection with abacterium that expresses a Group B streptococcal Ema polypeptidecomprising administering an immunogenically effective dose of a vaccinecomprising an Ema polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE to a subject.

The present invention is directed to a method for treating infectionwith a bacterium that expresses a Group B streptococcal Ema polypeptidecomprising administering a therapeutically effective dose of apharmaceutical composition comprising an Ema polypeptide selected fromthe group of EmaA, EmaB, EmaC, EmaD and EmaE, and a pharmaceuticallyacceptable carrier to a subject.

The invention further provides a method for treating infection with abacterium that expresses a Group B streptococcal Ema polypeptidecomprising administering a therapeutically effective dose of apharmaceutical composition comprising an antibody to an Ema polypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, and apharmaceutically acceptable carrier to a subject.

In a further aspect, the invention provides a method of inducing animmune response in a subject which has been exposed to or infected witha Group B streptococcal bacterium comprising administering to thesubject an amount of the pharmaceutical composition comprising an Emapolypeptide selected from the group of EmaA, EmaB, EmaC, EmaD and EmaE,and a pharmaceutically acceptable carrier, thereby inducing an immuneresponse.

The invention still further provides a method for preventing infectionby a streptococcal bacterium in a subject comprising administering tothe subject an amount of a pharmaceutical composition comprising anantibody to an Ema polypeptide selected from the group of EmaA, EmaB,EmaC, EmaD and EmaE and a pharmaceutically acceptable carrier ordiluent, thereby preventing infection by a streptococcal bacterium.

The invention further provides an ema mutant bacteria which isnon-adherent and/or non-invasive to cells and which is mutated in one ormore genes selected from the group of emaA, emaB, emaC, emaD and emaE.Particularly, such ema mutant is a Group B streptococcal bacteria. Suchnon-adherent and/or non-invasive ema mutant bacteria can further beutilized in expressing other immunogenic or therapeutic proteins for thepurposes of eliciting immune responses to any such other proteins in thecontext of vaccines and in other forms of therapy.

This invention provides a method of inhibiting colonization of hostcells in a subject which has been exposed to or infected with astreptococcal bacterium comprising administering to the subject anamount of a pharmaceutical composition comprising an Ema polypeptideselected from the group of EmaA, EmaB, EmaC, EmaD and EmaE, therebyinducing an immune response. The therapeutic peptide that blockscolonization is delivered by the respiratory mucosal. The pharmaceuticalcomposition comprises the polypeptide selected from the group of SEQ IDNO: 2, 4, 6, 8 and 10.

As used herein, “pharmaceutical composition” could mean therapeuticallyeffective amounts of polypeptide products or antibodies of the inventiontogether with suitable diluents, preservatives, solubilizers,emulsifiers, adjuvant and/or carriers useful in therapy againstbacterial infection or in inducing an immune response. A“therapeutically effective amount” as used herein refers to that amountwhich provides a therapeutic effect for a given condition andadministration regimen. Such compositions are liquids or lyophilized orotherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent absorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),solubilizing agents (e.g., glycerol, polyethylene glycerol),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives(e.g., Thimerosal, benzyl alcohol, parabens), bulking substances ortonicity modifiers (e.g., lactose, mannitol), covalent attachment ofpolymers such as polyethylene glycol to the protein, complexation withmetal ions, or incorporation of the material into or onto particulatepreparations of polymeric compounds such as polylactic acid, polglycolicacid, hydrogels, etc, or onto liposomes, microemulsions, micelles,unilamellar or multilamellar vesicles, erythrocyte ghosts, orspheroplasts. Such compositions will influence the physical state,solubility, stability, rate of in vivo release, and rate of in vivoclearance of the polypeptides of the present invention. The choice ofcompositions will depend on the physical and chemical properties of thepolypeptide. Controlled or sustained release compositions includeformulation in lipophilic depots (e.g., fatty acids, waxes, oils). Alsocomprehended by the invention are particulate compositions coated withpolymers (e.g., poloxamers or poloxamines) and the polypeptides of thepresent invention coupled to antibodies directed against tissue-specificreceptors, ligands or antigens or coupled to ligands of tissue-specificreceptors. Other embodiments of the compositions of the inventionincorporate particulate forms, protective coatings, protease inhibitorsor permeation enhancers for various routes of administration, includingparenteral, pulmonary, nasal and oral.

Further, as used herein “pharmaceutically acceptable carrier” are wellknown to those skilled in the art and include, but are not limited to,0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline.Additionally, such pharmaceutically acceptable carriers may be aqueousor non-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, antimicrobials, antioxidants, collating agents, inertgases and the like.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce by at least about 30percent, more preferably by at least 50 percent, most preferably by atleast 90 percent, a clinically significant infection by streptococcalbacterium. Alternatively, in the case of a vaccine or immunogeniccomposition, a therapeutically effective amount is used herein to meanan amount sufficient and suitable to elicit an immune response andantibody response in an individual, and particularly to provide aresponse sufficient to prevent, and preferably reduce by at least about30 percent, more preferably by at least 50 percent, most preferably byat least 90 percent, a clinically significant infection by streptococcalbacterium.

Controlled or sustained release compositions include formulation inlipophilic depots (e.g. fatty acids, waxes, oils). Also comprehended bythe invention are particulate compositions coated with polymers (e.g.poloxamers or poloxamines) and the compound coupled to antibodiesdirected against tissue-specific receptors, ligands or antigens orcoupled to ligands of tissue-specific receptors. Other embodiments ofthe compositions of the invention incorporate particulate formsprotective coatings, protease inhibitors or permeation enhancers forvarious routes of administration, including parenteral, pulmonary, nasaland oral.

When administered, compounds are often cleared rapidly from mucosalsurfaces or the circulation and may therefore elicit relativelyshort-lived pharmacological activity. Consequently, frequentadministrations of relatively large doses of bioactive compounds may byrequired to sustain therapeutic efficacy. Compounds modified by thecovalent attachment of water-soluble polymers such as polyethyleneglycol, copolymers of polyethylene glycol and polypropylene glycol,carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinylpyrrolidone or polyproline are known to exhibit substantiallylonger half-lives in blood following intravenous injection than do thecorresponding unmodified compounds (Abuchowski et al., 1981; Newmark etal., 1982; and Katre et al., 1987). Such modifications may also increasethe compound's solubility in aqueous solution, eliminate aggregation,enhance the physical and chemical stability of the compound, and greatlyreduce the immunogenicity and reactivity of the compound. As a result,the desired in vivo biological activity may be achieved by theadministration of such polymer-compound abducts less frequently or inlower doses than with the unmodified compound.

Dosages. The sufficient amount may include but is not limited to fromabout 1 .mu.g/kg to about 1000 mg/kg. The amount may be 10 mg/kg. Thepharmaceutically acceptable form of the composition includes apharmaceutically acceptable carrier.

As noted above, the present invention provides therapeutic compositionscomprising pharmaceutical compositions comprising vectors, vaccines,polypeptides, nucleic acids and antibodies, anti-antibodies, and agents,to compete with the Group B streptococcus bacterium for pathogenicactivities, such as adherence to host cells.

The preparation of therapeutic compositions which contain an activecomponent is well understood in the art. Typically, such compositionsare prepared as an aerosol of the polypeptide delivered to thenasopharynx or as injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, pH buffering agents which enhance the effectivenessof the active ingredient.

An active component can be formulated into the therapeutic compositionas neutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts (formed with the freeamino groups of the polypeptide or antibody molecule) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed from the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

A composition comprising “A” (where “A” is a single protein, DNAmolecule, vector, etc.) is substantially free of “B” (where “B”comprises one or more contaminating proteins, DNA molecules, vectors,etc.) when at least about 75% by weight of the proteins, DNA, vectors(depending on the category of species to which A and B belong) in thecomposition is “A”. Preferably, “A” comprises at least about 90% byweight of the A+B species in the composition, most preferably at leastabout 99% by weight.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to reduce by at least about 15 percent, preferably byat least 50 percent, more preferably by at least 90 percent, and mostpreferably prevent, a clinically significant deficit in the activity,function and response of the host. Alternatively, a therapeuticallyeffective amount is sufficient to cause an improvement in a clinicallysignificant condition in the host. In the context of the presentinvention, a deficit in the response of the host is evidenced bycontinuing or spreading bacterial infection. An improvement in aclinically significant condition in the host includes a decrease inbacterial load, clearance of bacteria from colonized host cells,reduction in fever or inflammation associated with infection, or areduction in any symptom associated with the bacterial infection.

According to the invention, the component or components of a therapeuticcomposition of the invention may be introduced parenterally,transmucosally, e.g., orally, nasally, pulmonarailly, or rectally, ortransdermally. Preferably, administration is parenteral, e.g., viaintravenous injection, and also including, but is not limited to,intra-arteriole, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial administration. Oralor pulmonary delivery may be preferred to activate mucosal immunity;since Group B streptococci generally colonize the nasopharyngeal andpulmonary mucosa, particularly that of neonates, mucosal immunity may bea particularly effective preventive treatment. The term “unit dose” whenused in reference to a therapeutic composition of the present inventionrefers to physically discrete units suitable as unitary dosage forhumans, each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectiozis Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In yet another embodiment, the therapeutic compound can be delivered ina controlled release system. For example, the polypeptide may beadministered using intravenous infusion, an implantable osmotic pump, atransdermal patch, liposomes, or other modes of administration. In oneembodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit.Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,polymeric materials can be used (see Medical Applications of ControlledRelease, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);Controlled Drug Bioavailability, Drug Product Design and Performance,Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, J.Macronol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al.,Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989);Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment,a controlled release system can be placed in proximity of thetherapeutic target, i.e., the brain, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release, supra, vol. 2, pp. 115-138 (1984)). Preferably, acontrolled release device is introduced into a subject in proximity ofthe site of inappropriate immune activation or a tumor. Other controlledrelease systems are discussed in the review by Langer (Science249:1527-1533 (1990)).

A subject in whom administration of an active component as set forthabove is an effective therapeutic regimen for a bacterial infection ispreferably a human, but can be any animal. Thus, as can be readilyappreciated by one of ordinary skill in the art, the methods andpharmaceutical compositions of the present invention are particularlysuited to administration to any animal, particularly a mammal, andincluding, but by no means limited to, domestic animals, such as felineor canine subjects, farm animals, such as but not limited to bovine,equine, caprine, ovine, and porcine subjects, wild animals (whether inthe wild or in a zoological garden), research animals, such as mice,rats, rabbits, goats, sheep, pigs, dogs, cats, etc., i.e., forveterinary medical use.

In the therapeutic methods and compositions of the invention, atherapeutically effective dosage of the active component is provided. Atherapeutically effective dosage can be determined by the ordinaryskilled medical worker based on patient characteristics (age, weight,sex, condition, complications, other diseases, etc.), as is well knownin the art. Furthermore, as further routine studies are conducted, morespecific information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age and generalhealth of the recipient, is able to ascertain proper dosing. Generally,for intravenous injection or infusion, dosage may be lower than forintraperitoneal, intramuscular, or other route of administration. Thedosing schedule may vary, depending on the circulation half-life, andthe formulation used. The compositions are administered in a mannercompatible with the dosage formulation in the therapeutically effectiveamount. Precise amounts of active ingredient required to be administereddepend on the judgment of the practitioner and are peculiar to eachindividual. However, suitable dosages may range from about 0.1 to 20,preferably about 0.5 to about 10, and more preferably one to several,milligrams of active ingredient per kilogram body weight of individualper day and depend on the route of administration. Suitable regimes forinitial administration and booster shots are also variable, but aretypified by an initial administration followed by repeated doses at oneor more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations of ten nanomolar to ten micromolarin the blood are contemplated.

Administration with other compounds. For treatment of a bacterialinfection, one may administer the present active component inconjunction with one or more pharmaceutical compositions used fortreating bacterial infection, including but not limited to (1)antibiotics; (2) soluble carbohydrate inhibitors of bacterial adhesin;(3) other small molecule inhibitors of bacterial adhesin; (4) inhibitorsof bacterial metabolism, transport, or transformation; (5) stimulatorsof bacterial lysis, or (6) anti-bacterial antibodies or vaccinesdirected at other bacterial antigens. Other potential active componentsinclude anti-inflammatory agents, such as steroids and non-steroidalanti-inflammatory drugs. Administration may be simultaneous (forexample, administration of a mixture of the present active component andan antibiotic), or may be in seriatim.

Accordingly, in specific embodiment, the therapeutic compositions mayfurther include an effective amount of the active component, and one ormore of the following active ingredients: an antibiotic, a steroid, etc.

Thus, in a specific instance where it is desired to reduce or inhibitthe infection resulting from a bacterium mediated binding of bacteria toa host cell, or an antibody thereto, or a ligand thereof or an antibodyto that ligand, the polypeptide is introduced to block the interactionof the bacteria with the host cell.

Also contemplated herein is pulmonary delivery of an inhibitor of thepolypeptide of the present invention having which acts as adhesininhibitory agent (or derivatives thereof). The adhesin inhibitory agent(or derivative) is delivered to the lungs of a mammal, where it caninterfere with bacterial, i.e., streptococcal, and preferably Group Bstreptococcal binding to host cells. Other reports of preparation ofproteins for pulmonary delivery are found in the art [Adjei et al.(1990) Pharmaceutical Research, 7:565-569; Adjei et al. (1990)International Journal of Pharmaceutics, 63:135-144 (leuprolide acetate);Braquet et al (1989), Journal of Cardiovascular Pharmacology, 13(suppl.5):143-146 (endothelin-1); Hubbard et al. (1989) Annals of InternalMedicine, Vol. III, pp. 206-212 (.alpha.-1-antitrypsin); Smith et al.(1989) J. Clin. Invest. 84:1145-1146 (.alpha.-1-proteinase); Oswein etal., “Aerosolization of Proteins”, Proceedings of Symposium onRespiratory Drug Delivery II, Keystone, Colo., March, (1990)(recombinant human growth hormone); Debs et al. (1988) J. Immunol.140:3482-3488 (interferon-.gamma. and tumor necrosis factor alpha);Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulatingfactor)]. A method and composition for pulmonary delivery of drugs isdescribed in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong etal.

All such devices require the use of formulations suitable for thedispensing of adhesin inhibitory agent (or derivative). Typically, eachformulation is specific to the type of device employed and may involvethe use of an appropriate propellant material, in addition to the usualdiluents, adjuvant and/or carriers useful in therapy. Also, the use ofliposomes, microcapsules or microspheres, inclusion complexes, or othertypes of carriers is contemplated. Chemically modified adhesininhibitory agent may also be prepared in different formulationsdepending on the type of chemical modification or the type of deviceemployed.

Formulations suitable for use with a, nebulizer, either jet orultrasonic, will typically comprise adhesin inhibitory agent (orderivative) dissolved in water at a concentration of about 0.1 to 25 mgof biologically active adhesin inhibitory agent per ml of solution. Theformulation may also include a buffer and a simple sugar (e.g., foradhesin inhibitory agent stabilization and regulation of osmoticpressure). The nebulizer formulation may also contain a surfactant, toreduce or prevent surface induced aggregation of the adhesin inhibitoryagent caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the adhesin inhibitory agent(or derivative) suspended in a propellant with the aid of a surfactant.The propellant may be any conventional material employed for thispurpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol, and1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactantsinclude sorbitan trioleate and soya lecithin. Oleic acid may also beuseful as a surfactant.

The liquid aerosol formulations contain adhesin inhibitory agent and adispersing agent in a physiologically acceptable diluent. The dry powderaerosol formulations of the present invention consist of a finelydivided solid form of adhesin inhibitory agent and a dispersing agent.With either the liquid or dry powder aerosol formulation, theformulation must be aerosolized. That is, it must be broken down intoliquid or solid particles in order to ensure that the aerosolized doseactually reaches the mucous membranes of the nasal passages or the lung.The term “aerosol particle” is used herein to describe the liquid orsolid particle suitable for nasal or pulmonary administration, i.e.,that will reach the mucous membranes. Other considerations, such asconstruction of the delivery device, additional components in theformulation, and particle characteristics are important. These aspectsof pulmonary administration of a drug are well known in the art, andmanipulation of formulations, aerosolization means and construction of adelivery device require at most routine experimentation by one ofordinary skill in the art. In a particular embodiment, the mass mediandynamic diameter will be 5 micrometers or less in order to ensure thatthe drug particles reach the lung alveoli [Wearley, L. L. (1991) Crit.Rev. in Ther. Drug Carrier Systems 8:333].

Systems of aerosol delivery, such as the pressurized metered doseinhaler and the dry powder inhaler are disclosed in Newman, S. P.,Aerosols and the Lung, Clarke, S. W. and Davia, D. editors, pp. 197-22and can be used in connection with the present invention.

In a further embodiment, as discussed in detail infra, an aerosolformulation of the present invention can include other therapeuticallyor pharmacologically active ingredients in addition to adhesininhibitory agent, such as but not limited to an antibiotic, a steroid, anon-steroidal anti-inflammatory drug, etc.

Liquid Aerosol Formulations. The present invention provides aerosolformulations and dosage forms for use in treating subjects sufferingfrom bacterial, e.g., streptococcal, in particularly streptococcal,infection. In general such dosage forms contain adhesin inhibitory agentin a pharmaceutically acceptable diluent. Pharmaceutically acceptablediluents include but are not limited to sterile water, saline, bufferedsaline, dextrose solution, and the like. In a specific embodiment, adiluent that may be used in the present invention or the pharmaceuticalformulation of the present invention is phosphate buffered saline, or abuffered saline solution generally between the pH 7.0-8.0 range, orwater.

The liquid aerosol formulation of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, surfactants and excipients. Theformulation may include a carrier. The carrier is a macromolecule whichis soluble in the circulatory system and which is physiologicallyacceptable where physiological acceptance means that those of skill inthe art would accept injection of said carrier into a patient as part ofa therapeutic regime. The carrier preferably is relatively stable in thecirculatory system with an acceptable plasma half life for clearance.Such macromolecules include but are not limited to Soya lecithin, oleicacid and sorbitan trioleate, with sorbitan trioleate preferred.

The formulations of the present embodiment may also include other agentsuseful for pH maintenance, solution stabilization, or for the regulationof osmotic pressure. Examples of the agents include but are not limitedto salts, such as sodium chloride, or potassium chloride, andcarbohydrates, such as glucose, galactose or mannose, and the like.

The present invention further contemplates liquid aerosol formulationscomprising adhesin inhibitory agent and another therapeuticallyeffective drug, such as an antibiotic, a steroid, a non-steroidalanti-inflammatory drug, etc.

Aerosol Dry Powder Formulations. It is also contemplated that thepresent aerosol formulation can be prepared as a dry powder formulationcomprising a finely divided powder form of adhesin inhibitory agent anda dispersant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing adhesin inhibitory agent (orderivative) and may also include a bulking agent, such as lactose,sorbitol, sucrose, or mannitol in amounts which facilitate dispersal ofthe powder from the device, e.g., 50 to 90% by weight of theformulation. The adhesin inhibitory agent (or derivative) should mostadvantageously be prepared in particulate form with an average particlesize of less than 10 mm (or microns), most preferably 0.5 to 5 mm, formost effective delivery to the distal lung. In another embodiment, thedry powder formulation can comprise a finely divided dry powdercontaining adhesin inhibitory agent, a dispersing agent and also abulking agent. Bulking agents useful in conjunction with the presentformulation include such agents as lactose, sorbitol, sucrose, ormannitol, in amounts that facilitate the dispersal of the powder fromthe device.

The present invention further contemplates dry powder formulationscomprising adhesin inhibitory agent and another therapeuticallyeffective drug, such as an antibiotic, a steroid, a non-steroidalanti-inflammatory drug, etc.

Contemplated for use herein are oral solid dosage forms, which aredescribed generally in Remington's Pharmaceutical Sciences, 18th Ed.1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which isherein incorporated by reference. Solid dosage forms include tablets,capsules, pills, troches or lozenges, cachets or pellets. Also,liposomal or proteinoid encapsulation may be used to formulate thepresent compositions (as, for example, proteinoid microspheres reportedin U.S. Pat. No. 4,925,673). Liposomal encapsulation may be used and theliposomes may be derivatized with various polymers (e.g., U.S. Pat. No.5,013,556). A description of possible solid dosage forms for thetherapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited byG. S. Banker and C. T. Rhodes Chapter 10, 1979, herein incorporated byreference. In general, the formulation will include the component orcomponents (or chemically modified forms thereof) and inert ingredientswhich allow for protection against the stomach environment, and releaseof the biologically active material in the intestine.

Also specifically contemplated are oral dosage forms of the abovederivatized component or components. The component or components may bechemically modified so that oral delivery of the derivative isefficacious. Generally, the chemical modification contemplated is theattachment of at least one moiety to the component molecule itself,where said moiety permits (a) inhibition of proteolysis; and (b) uptakeinto the blood stream from the stomach or intestine. Also desired is theincrease in overall stability of the component or components andincrease in circulation time in the body. Examples of such moietiesinclude: polyethylene glycol, copolymers of ethylene glycol andpropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981,“Soluble Polymer-Enzyme Abducts” In: Enzymes as Drugs, Hocenberg andRoberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383; Newmark,et al. (1982) J. Appl. Biochem. 4:185-189. Other polymers that could beused are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred forpharmaceutical usage, as indicated above, are polyethylene glycolmoieties.

For the component (or derivative) the location of release may be thestomach, the small intestine (the duodenum, the jejunem, or the ileum),or the large intestine. One skilled in the art has availableformulations which will not dissolve in the stomach, yet will releasethe material in the duodenum or elsewhere in the intestine. Preferably,the release will avoid the deleterious effects of the stomachenvironment, either by protection of the protein (or derivative) or byrelease of the biologically active material beyond the stomachenvironment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH5.0 is essential. Examples of the more common inert ingredients that areused as enteric coatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), BPMCP 50, BPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which arenot intended for protection against the stomach. This can include sugarcoatings, or coatings which make the tablet easier to swallow. Capsulesmay consist of a hard shell (such as gelatin) for delivery of drytherapeutic i.e. powder; for liquid forms, a soft gelatin shell may beused. The shell material of cachets could be thick starch or otheredible paper. For pills, lozenges, molded tablets or tablet triturates,moist massing techniques can be used.

The peptide therapeutic can be included in the formulation as finemultiparticulates in the form of granules or pellets of particle sizeabout 1 mm. The formulation of the material for capsule administrationcould also be as a powder, lightly compressed plugs or even as tablets.The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, theprotein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the therapeutic with an inertmaterial. These diluents could include carbohydrates, especiallymannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modifieddextran and starch. Certain inorganic salts may be also be used asfillers including calcium triphosphate, magnesium carbonate and sodiumchloride. Some commercially available diluents are Fast-Flo, Emdex,STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrates include but are notlimited to starch, including the commercial disintegrant based onstarch, Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants. Binders may be used to hold the therapeutic agenttogether to form a hard tablet and include materials from naturalproducts such as acacia, tragacanth, starch and gelatin. Others includemethyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose(CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose(HPMC) could both be used in alcoholic solutions to granulate thetherapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium auryl sulfate, magnesium lauryl sulfate,polyethylene glycol of various molecular eights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment asurfactant might be added as a wetting agent. Surfactants may includeanionic detergents such as sodium lauryl sulfate, dioctyl sodiumsulfosuccinate and dioctyl sodium sulfonate. Cationic detergents mightbe used and could include benzalkonium chloride or benzethomiumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives which potentially enhance uptake of the polypeptide (orderivative) are for instance the fatty acids oleic acid, linoleic acidand linolenic acid.

Pulmonary Delivery. Also contemplated herein is pulmonary delivery ofthe present polypeptide (or derivatives thereof). The polypeptide (orderivative) is delivered to the lungs of a mammal while inhaling andcoats the mucosal surface of the alveoli. Other reports of this includeAdjei et al. (1990) Pharmaceutical Research 7:565-569; Adjei et al.(1990) International Journal of Pharmaceutics 63:135-144 (leuprolideacetate); Braquet et al. (1989) Journal of Cardiovascular Pharmacology,13 (suppl. 5):143-146 (endothelin-1); Hubbard et al. (1989) Annals ofInternal Medicine, Vol. III, pp. 206-212 (a1-antitrypsin); Smith et al.(1989) J. Clin. Invest. 84:1145-1146 (a-1-proteinase); Oswein et al.(1990) “Aerosolization of Proteins”, Proceedings of Symposium onRespiratory Drug Delivery II, Keystone, Colo., March, (recombinant humangrowth hormone); Debs et al. (1988) J. Immunol. 140:3482-3488(interferon-g and tumor necrosis factor alpha) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor). A method andcomposition for pulmonary delivery of drugs for systemic effect isdescribed in U.S. Pat. No. 5,451,569, issued Sep. 19, 1995 to Wong etal.

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art.

Formulations suitable for use with a, nebulizer, either jet orultrasonic, will typically comprise polypeptide (or derivative)dissolved in water at a concentration of about 0.1 to 25 mg ofbiologically active protein per mL of solution. The formulation may alsoinclude a buffer and a simple sugar (e.g., for protein stabilization andregulation of osmotic pressure). The nebulizer formulation may alsocontain a surfactant, to reduce or prevent surface induced aggregationof the protein caused by atomization of the solution in forming theaerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the polypeptide (orderivative) suspended in a propellant with the aid of a surfactant. Thepropellant may be any conventional material employed for this purpose,such as a chlorofluorocarbon, a hydrochlorofluorocarbon, ahydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane,dichlorodifluoromethane, dichlorotetrafluoroethanol and1,1,1,2-tetrafiluoroethane, or combinations thereof. Suitablesurfactants include sorbitan trioleate and soya lecithin. Oleic acid mayalso be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing polypeptide (or derivative) and mayalso include a bulking agent, such as lactose, sorbitol, sucrose, ormannitol in amounts which facilitate dispersal of the powder from thedevice, e.g., 50 to 90% by weight of the formulation. The protein (orderivative) should most advantageously be prepared in particulate formwith an average particle size of less than 10 mm (or microns), mostpreferably 0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal Delivery. Nasal or nasopharyngeal delivery of the polypeptide (orderivative) is also contemplated. Nasal delivery allows the passage ofthe polypeptide directly over the upper respiratory tract mucosal afteradministering the therapeutic product to the nose, without the necessityfor deposition of the product in the lung. Formulations for nasaldelivery include those with dextran or cyclodextran, tide nomenclature,J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid

The therapeutic polypeptide-, analog- or active fragment-containingcompositions are conventionally administered intravenously, as byinjection of a unit dose, for example. The term “unit dose” when used inreference to a therapeutic composition of the present invention refersto physically discrete units suitable as unitary dosage for humans, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect in association with therequired diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofinhibition or neutralization of about binding capacity desired. Preciseamounts of active ingredient required to be administered depend on thejudgment of the practitioner and are peculiar to each individual.However, suitable dosages may range from about 0.1 to 20, preferablyabout 0.5 to about 10, and more preferably one to several, milligrams ofactive ingredient per kilogram body weight of individual per day anddepend on the route of administration. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by repeated doses at one or more hourintervals by a subsequent injection or other administration.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations of ten nanomolar to ten micromolar in the blood arecontemplated.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

Example 1 Identification of Group B Streptococcus Genes

Comparing the genetic and phenotypic composition of genetically-relatedgroups of a bacterial species facilitates identifying virulence factorspresent in the most pathogenic groups. Type III GBS can be subdividedinto three groups of related strains based on the analysis ofrestriction digest patterns (RDPs) produced by digestion of chromosomalDNA with Hind III and Sse 8387 (5, 6). Over 90% of invasive type III GBSdisease in neonates in Japan and in Salt Lake City is caused by bacteriafrom one of three RDP types, termed RDP type III-3, while RDP type III-2are significantly more likely to be isolated from vagina than from bloodor CSF (6). These results suggest that this genetically-related clusterof type III-3 GBS are more virulent than III-2 strains and could beresponsible for the majority of invasive type III disease globally. Weproposed that bacterial factors that contribute to the increasedvirulence of III-3 strains can be identified by characterizing thedifferences between the genetic composition of III-3 and III-2 strains.Such genetic differences will be found in the bacterial chromosomessince these strains do not contain plasmids (6).

To identify genes present in virulent type III-3 GBS strains and not inthe avirulent type III-2 strains we used a modification of the techniquedescribed by Lisitsyn et al (7). High molecular weight genomic DNA froman invasive RDP type III-3 GBS strain (strain 874391) and a colonizing(“avirulent”) RDP type III-2 strain (strain 865043) was prepared by celllysis with mutanolysin and Proteinase K digestion (5). For geneticsubtraction, genomic DNA from both strains was digested with Taq I. TaqI-digested DNA from the virulent strain was mixed with two complementaryoligonucleotides (TaqA (5′-CTAGGTGGATCCTTCGGCAAT-3′ (SEQ ID NO: 11)) andTaqB (5′-CGATTGCCGA-3′ (SEQ ID NO: 12)), heated to 50.degree. C. for 5minutes, then allowed to cool slowly to 16.degree. C. in T4 ligasebuffer. Oligonucleotides were ligated to the virulent strain DNA byincubation with 20 units of T4 ligase at 16.degree. C. for 12 hours.After ligation, 500 ng of DNA from the virulent strain, with ligatedlinkers, and 40 ug of DNA from the avirulent strain, without linkers,was mixed together, denatured by heating, and hybridized at 68.degree.C. for 20 hours.

Ten percent of the resulting hybridization mixture was incubated withTaq DNA polymerase and dNTPs to fill in the ends of annealed virulentstrain DNA. The hybridized DNA was amplified by Taq DNA polymerase for10 cycles using the TaqA oligonucleotide as the forward and reverseamplification primer. After amplification, single stranded productsremaining after amplification were digested with mung bean nuclease.Twenty percent of the resulting product was then reamplified for 20cycles. This process of subtraction followed by PCR amplificationresults in enhanced amplification of DNA segments from the III-3 strainsthat do not hybridize with DNA segments from the III-2 strains.

A total of four cycles of subtraction and amplification were carriedout, using successively smaller quantities of III-3 specific PCRproducts and alternating two sets of adaptors (TaqA/B (SEQ ID NOS: 11and 12, respectively) and TaqE/F (TaqE (5′-AGGCAACTGTGCTAACCGAGGGAAT-3′(SEQ ID NO: 13)); and TaqF (5′-CGATTCCCTCG-3′ (SEQ ID NO: 14)). Thefinal amplification products were ligated into pBS KS+ vectors. Thirteenclones were randomly selected for analysis. These probes were used inslot and dot blot experiments to determine whether subtraction wassuccessful and to identify probes hybridizing with all III-3 strains.Each of the 6 unique probes hybridized with the parental III-3 virulentstrain, while none of the probes hybridized with the avirulent III-2strains. Two of the amplified sequence tags (clones DY1-1 and DY1-11)hybridized with genomic DNA from all 62 type III isolates, but did nothybridize with DNA prepared from the III-2 and III-1 isolates (FIG. 1).To obtain additional sequence information, we constructed a genomic GBSIII-3 library. Multiple plaques hybridizing with each of the III-3GBS-specific probes have been purified for further characterization.

Results

The spb Locus

Three overlapping genomic clones hybridizing with probe DY1-1 wereidentified. A 6.4 kb Sal I-Bgl II fragment present in each clone wassubcloned and sequenced. This genomic DNA is present in all RDP typeIII-3 strains but not in serotype III-2, III-1 or other GBS serotypestrains.

Over 90% of this genomic DNA fragment has been sequenced and found tocontain 5 open reading frames (ORFs). Two ORFs appear to be candidatesfor virulence genes. spb1 is a 1509 by ORF. The predicted protein (502amino acids and Mr 53,446) has the characteristics of a cell-wall boundprotein. The nucleic acid and predicted amino acid sequences of sbp1 areprovided, in SEQ ID NOS: 15 and 16, respectively. The N-terminus of thepredicted protein is a hydrophilic, basic stretch of 6 amino acidsfollowed by a 23 amino acid hydrophobic, proline-rich core, consistentwith a signal peptide. The hydrophilic mature protein terminates in atypical LPXTG (SEQ ID NO: 17) domain that immediately precedes ahydrophobic 20 amino acid core and a short, basic hydrophilic terminus.The nucleotide sequence is not homologous to sequences of other knownbacterial genes. The translated amino acid sequence, however, sharessegmental homology with a number of characterized proteins, includingthe fimbrial type 2 protein of Actinomyces naeslundii (27% identity over350 amino acids) and the fimbrial type I protein of Actinomyces viscosus(25% homology over 420 amino acids) (16), the T6 surface protein of S.pyogenes (23% identity over 359 amino acids) (20), and the hsf (27%identity over 260 amino acids) and HMW1 adhesins (25% identity over 285amino acids) of Haemophilus influenzae (21, 22). The function of the S.pyogenes T6 protein is unknown. Each of the other homologs plays a rolein bacterial adhesion and/or invasion.

A spb1.sup.-isogenic deletion mutant GBS strain was created byhomologous recombination (using the method as described in Example 2below) and the ability of the spb1.sup.-mutant to adhere to and invadeA549 respiratory epithelial cells was determined. Compared to the wildtype strain, the number of spb1 .sup.-bacteria adherent to A549monolayers was reduced by 60.0% (p<0.01) and the number of intracellularinvading bacteria was reduced by 53.6% (p<0.01). This data suggests spb1may contribute to the pathogenesis of GBS pneumonia and bacterial entryinto the bloodstream.

The second ORF, spb2, terminates 37 bp upstream from spb1 and is in thesame transcriptional orientation. This 1692 bp ORF has a deduced aminoacid sequence of 579 residues and Mr 64,492. The nucleic acid andpredicted amino acid sequences of sbp2 are provided in SEQ ID NOS: 18and 19, respectively. spb2 shares 50.5% nucleic acid identity and 20.7%amino acid identity with spb1. Conservation is highest in thecarboxy-terminal regions, including a shared LPSTGG (SEQ ID NO: 20)motif. In contrast to spb1, spb2 does not have a obvious signalsequence. Its secretion may be mediated by carboxy-terminal recognitionsequences or by accessory peptides (23). The deduced amino acid sequenceof Spb2 is also homologous with S. pyogenes T6 and Actinomycesnaeslundii proteins, and to Listeria monocytogenes internalin A (22%identity over 308 amino acids); again, proteins important in adhesionand invasion (24).

The ema Locus

Two genomic clones hybridizing with probe DY1-11 were identified. A 7 kbHind III fragment present in each clone was subcloned and sequenced.Unlike the serotype III specific spb sequences, this genomic DNA, whichis adjacent to a region of serotype III-3 specific DNA, was found to bepresent in all GBS tested to date, including serotype Ia, Ib, II and Vstrains. This region of the GBS chromosome, which we have designated theextracellular matrix adhesin (ema) locus, contains 5 significant ORFs.

emaA

The first ORF, emaA, is 738 bp long, with a predicted protein product of246 amino acids and Mr 26.2. The nucleic acid sequence (SEQ ID NO: 1)and predicted amino acid sequence (SEQ ID NO: 2) of emaA are shown inFIG. 2. The EmaA protein is a non-repetitive protein. The 27 amino acidN-terminus of the predicted protein is consistent with a signal peptide.The mature protein has an imperfect cell wall binding domain (XPXTGG(SEQ ID NO:21)) followed by a transmembrane spanning domain encompassingresidues 219-235 and a terminal hydrophilic tail. The emaA nucleotidesequence is not homologous to known sequences of bacterial genes. Thetranslated amino acid sequence, however, shares segmental homology witha number of characterized proteins, including a collagen adhesin, Bbp,of Staphylococcus aureus (37% identity over 103 aa) (15), a type 2fimbrial structural subunit of Actinomyces naeslandii (39% homology over112 aa) (16), and the FimP protein of Actinomyces viscosus (28% homologyover 228 aa) (17). The function of the S. pyogenes T6 protein isunknown. The type 1 and type 2 fimbria of Actinomyces mediate bacterialadhesion to salivary glycoproteins and various host cells, contributingto the pathogenesis of dental caries.

emaB

The second ORF, emaB, begins 94 bp 3′ of emaA and is in the sametranscriptional orientation. The nucleic acid sequence (SEQ ID NO: 3)and predicted amino acid sequence (SEQ ID NO: 4) of emaB are shown inFIG. 3. It is 924 bp long with a predicted protein product of 308 aminoacids and Mr 33.9. The predicted EmaB protein is a nonrepetitiveprotein. The 27 amino acids N-terminus of the predicted protein isconsistent with a signal peptide. The mature protein has an imperfectcell wall binding domain (XPXTG) followed by a transmembrane spanningdomain encompassing residues 279-294. The enaB nucleotide sequence isnot homologous to known sequences of bacterial genes. The translatedamino acid sequence, however, shares segmental homology with a number ofcharacterized proteins, including a type 2 fimbrial structural subunitof Actinomyces naeslandii (28% homology over 222 amino acids), the T6protein of S. pyogenes (26% homology over 266 amino acids) (20), and aS. epidermidis putative cell-surface adhesin (24% identity over 197amino acids). The first of these proteins mediates adhesion of S. aureusto collagen and is postulated to contribute to the pathogenesis ofosteomyelitis and infectious arthritis.

emaC

The third ORF, emaC, begins 2 bp 3′ of emaB and is the sametranscriptional orientation. It is 918 bp long, with a predicted proteinproduct of 305 amino acids and Mr 34.5. The nucleic acid sequence (SEQID NO: 5) and predicted amino acid sequence (SEQ ID NO: 6) of emaC aredepicted in FIG. 4. The EmaC protein is a nonrepetitive protein. The 30amino acid N-terminus of the predicted protein is consistent with asignal peptide. The mature protein has a transmembrane spanning domainencompassing residues 265-281. The emaC nucleotide sequence is nothomologous to known sequences of bacterial genes. The translated aminoacid sequence, however, shares segmental homology with a number ofcharacterized proteins, including proteins associated with the assemblyof type 2 fimbrial structural subunit of Actinomyces naeslandii (38%homology over 234 amino acids) (16). These proteins are required for theassembly of type 2 fimbria.

emaD

The fourth ORF, emaD, is 852 bp long, overlaps emaC by 47 bp, and is inthe same transcriptional orientation. The predicted protein product is284 amino acids and Mr 33.1. The nucleic acid sequence (SEQ ID NO: 7)and predicted amino acid sequence (SEQ ID NO:8) of emaD are shown inFIG. 5. No indentifiable N-terminal signal sequence is present andpotential transmembrane segments are present at positions 19-35 and252-280. The mature protein is not repetitive and lacks a cell wallbinding domain. The emaD nucleotide sequence is not homologous to knownsequences of bacterial genes. The translated amino acid sequence, sharessegmental homology with the same fimbria-associated proteins ofActinomyces as does EmaC.

emaE

The fifth ORF, emaE, begins 42 bp 3′ of emaD and is in the sametranscriptional orientation. It is 2712 bp long, with a predictedprotein product of 904 aa and Mr 100.9. FIG. 6 depicts the nucleic acidsequence (SEQ ID NO: 9) and predicted amino acid sequence (SEQ ID NO:10) of emaE. The predicted EmaE protein is a nonrepetitive protein. Anobvious N-terminal signal peptide is not evident but a putativetransmembrane region is located at residues 24-40. The mature proteinhas an imperfect cell wall binding domain (XPXTGG (SEQ ID NO: 21))followed by a transmembrane spanning domain encompassing residues880-896. The emaE nucleotide sequence is not homologous to knownsequences of bacterial genes. The translated amino acid sequence,however, shares segmental homology with a number of characterizedproteins, including the F1 and F2 fibronectin binding proteins of S.pyogenes (31% homology over 207 amino acids) (18, 19). These proteinsmediate high affinity binding to fibronectin, and are important in theadhesion of S. pyogenes to respiratory cells.

The similarity of the protein products of the ema locus tophysiologically important adhesins and invasins of other bacterialspecies suggests that the Ema proteins have a role in facilitating theadhesion of GBS to extracellular matrix components and to cell surfacesand subsequent invasion of epithelial and endothelial cells, the initialsteps in the pathogenesis of infection.

Several lines of evidence suggest the members of the enia and the spblocus may have similar functions, but are likely to represent distinctclasses of proteins. The ema and spb locus genes are each and allsimilar to physiologically important adhesions and invasions of thebacterial species, however, both Spb1 and Spb2 have prototypical grampositive cell-wall binding domains, whereas the members of the ema locushave an unusual motif, suggesting a distinct mechanism of cell surfaceanchoring. Second, the spb locus is restricted to virulent serotypeIII-3 strains of GBS, whereas the ema locus appears to be ubiquitous inall GBS serotypes. Third, spb1 and spb2 are more homologous to oneanother than to members of the ema locus and ema genes are more closelyhomologous to one another than to spb1 and spb2.

Example 2 Biologic Characterization of Novel GBS Genes

Isogenic Mutant Bacterial Strains

To identify biologic activity of these novel GBS genes, isogenic mutantbacterial strains are created which are identical in all respects exceptfor the presence or absence of a particular gene. Deletion mutants arecreated by allelic replacement. The relevant gene, with 100-300 bp offlanking sequences, is subcloned and modified by the deletion of anintragenic portion of the coding sequence and, in some cases, theinsertion of a kanamycin resistance gene. The mutant gene is cloned intothe suicide vector pHY304 (kindly provided by Dr. Craig Rubens), a broadhost range plasmid containing a temperature sensitive ori, erythromycinresistance gene (erm.sup.TS), and a pBS multiple cloning site. ThepHY304 vector is a derivative of the vector pWV01 (Framson, P. E. et al(1997) Applied Environ Microbiology 63:3539-3547). Plasmids containingmutant genes are electroporated into strain 874391 and single cross-overmutants are selected by antibiotic resistance at 37.degree. C. Theresulting antibiotic resistant colonies are subjected to a temperatureshift to 30.degree. C. Integration of the plasmid is unstable at thispermissive temperature because there are two functional ori's on thechromosome. Excised plasmid is eliminated by growth on nonselectivemedia for many generations, then colonies are screened for the presenceof the mutant allele by erythromycin-sensitivity. Double-crossovermutants are stable and do not require maintenance under drug selection.The mutant genotype is confirmed by Southern blotting or PCRdemonstrating the appropriate deletion. The resulting mutants arescreened for the presence of gene expression by Northern and Westernblot analysis. The phenotype of the knockout mutants is then comparedwith that of the wild type strain 874391 by examining growth rate andcolony morphology, and the expression of .beta.-hemolysin and CAMPfactor. Surface protein expression is assessed by Western blot, usingpolyclonal sera from rabbits immunized with whole, heat-killed type IIIGBS.

In Vitro Models

A. Adherence

Adhesion of GBS to host cells is a prerequisite for invasive disease.Three different cell types have the potential to be important in thisprocess: i) adhesion to respiratory epithelial cells is likely tofacilitate most early onset neonatal infections, ii) adhesion togastrointestinal epithelial cells has been postulated to be important inthe pathogenesis of late onset neonatal infections, and iii) adhesion toendothelial cells is necessary for both endocarditis and otherendovascular infections, and is likely to be the initial event in GBSmeningitis. The ability of wild type and mutant strains to adhere toepithelial and endothelial cells is compared in adhesion assays.

Four different cell lines are used to investigate the role of novel GBSgenes in adhesion. GBS adhere to and invade A549 human alveolarepithelial carcinoma cells and surface proteins appear to play animportant role in this process (8). GBS binding to A549 cells is used asan in vitro model for respiratory colonization. GBS also adhere toC2BBeL, a human intestinal epithelial cell line, which is used as amodel for gastrointestinal colonization, and to HeLa cervical epithelialcells, a model for genital colonization and maternal infection. Forendothelial adhesion, two cell lines are studied: freshly isolated humanumbilical vein endothelial (HUVE) cells; and an immortalized human brainmicrovascular endothelial cell line (BMEC). Adhesion assays areperformed as described by Tamura et al (9). Cell lines are grown toconfluence in 96-well tissue culture plates in recommended media.Monolayers are washed with PBS and fixed with 0.5% gluteraldehyde.Following blocking with 5% BSA in PBS, cells are inoculated with variousinocula of GBS, centrifuged for 10 minutes at 2000 rpm and incubated for1 hour at 4.degree. C. Nonadherent bacteria are removed by washing threetimes with 5% nonfat dry milk in PBS and bound bacteria are then elutedand plated quantitatively.

B. Invasion

GBS adhere to and invade respiratory epithelium, endothelium and BMEC(8, 10, 11). The ability of wild type and isogenic mutant GBS strains toinvade the above epithelial and endothelial cells are tested aspreviously described (8, 10, 11). Assays that distinguish the ability ofGBS to invade eukaryotic cells versus adhere to cells capitalize on theinability of penicillin and gentamicin to enter host cells, allowingquantification of intracellular bacteria after extracellular bacteriaare killed. GBS are grown to the desired growth phase in TH broth,washed twice with PBS and resuspended in tissue culture media containing10% fetal calf serum. Tissue culture monolayers grown to confluence in24-well plates are inoculated with varying inocula of GBS, centrifugedat 800×g and incubated at 37.degree. C. in 5% CO.sub.2 for 2-6 hours.Extracellular bacteria are removed by washing four times with PBS. Cellsare then incubated in fresh medium with 5 mg/ml penicillin and 100 mg/mlgentamicin for 2 hours. Media is then removed, monolayers washed, andcells lysed by treatment with 0.025% Triton X-100. Cell lysates aresonicated to disrupt bacterial chains and aliquots platedquantitatively.

C. Antibody to GBS Proteins

The ability of specific antibody to the novel GBS proteins to promoteopsonophagocytic killing of GBS is tested (12). Rabbits are immunizedwith recombinant or purified GBS proteins produced by standardtechniques. Rabbit antiserum of different dilutions (ranging from 1/50to 1/5,000) that has been exhaustively absorbed with the relevantisogenic mutant strain at 4.degree. C. will be incubated with GBS in thepresence of human complement and polymorphonuclear leukocytes(3.times.10.sup.6). Opsonophagocytic killing is expressed as the lognumber of CFU surviving following 1 hour of incubation subtracted fromthe log of the number of CFU at the zero time point. Killing of wildtype strains is compared to that of isogenic mutants lacking novel GBSproteins.

In Vivo Models

The neonatal rat has been used by numerous laboratories as a model ofGBS infection because it closely mimics human neonatal infection (13).The contribution of novel genes to the pathogenesis of GBS infections istested by comparing wild type and mutant in this system. Rat pups areinoculated by two routes. First, pups are inoculated intranasally tomimic the respiratory infection and sepsis typical of early onset GBSinfection. Secondly, intraperitoneal or subcutaneous inoculationreproduces the high grade bacteremia associated with GBS sepsis and thatprecedes GBS meningitis (14).

Rat pups are inoculated with varying doses of GBS strains and mortalityis determined. The level of bacteremia is determined by quantitativeblood cultures. Lung, liver, spleen and meningeal tissue are preservedfor histologic examination.

The ability of antiserum to the GBS proteins to protect neonatal ratsfrom GBS infection is tested (13). Newborn rats (<18 hours old) receivean intraperitoneal injection of 0.5 ml of undiluted rabbit antiserum,followed by the intraperitoneal inoculation of the equivalent of oneLD50 unit of GBS (usually about 5000 bacteria) in PBS. Mortality andmorbidity are then determined.

Role of Novel GBS Proteins in Vaccines

Several surface proteins of GBS, including C and Rib are immunogenic andprotective against GBS infection in infant rodent models (25, 26). Noneof these proteins are present in all GBS strains (27). Furthermore, eachof these proteins has a repetitive structure. The phenotypic variabilityof these repetitive proteins allows escape mutants expressing variantforms to evade host immune systems and may limit the effectiveness ofthese vaccines (28). It is notable that each of the predicted proteinsof the spb and ema loci do not have a repetitive structure and would nothave this disadvantage.

The novel GBS proteins we describe here may be useful antigens for a GBSvaccine. The data presented herein indicates these proteins have a rolein mediating adhesion to and invasion of GBS to human epithelial cells,thus antibody against these antigens may prevent these initial steps ininfection. It is highly desirable to develop a vaccine that preventscolonization of pregnant women and other individuals at increased riskof invasive GBS infection, as this would eliminate most infections. Ourdata suggests that antibody against Spb 1 is effective in reducingcolonization or infection following colonization with highly virulentstrains of serotype III, and therefore this protein is a particularlyuseful vaccine antigen. Members of the ema locus, unlike spb 1 and spb2,are ubiquitous in GBS and therefore have a role in the prevention ofinfection by multiple serotypes of GBS. An optimal vaccine formulationincludes combinations of these antigens.

Two strategies are used to design GBS vaccines using these novelproteins. First, purified recombinant or affinity-purified proteins areused as vaccine antigens, singly or in combination (25). Second, theseproteins are used as carrier proteins for capsular polysaccharide oroligosaccharide-based vaccines. GBS polysaccharides and oligosaccharidesare generally poorly immunogenic and fail to elicit significant memoryand booster responses (29). Conjugation of these polysaccharides oroligosaccharides to protein carriers increases immunogenicity. GBSpolysaccharide conjugated to tetanus toxoid, for example, has been usedto immunize pregnant women and results in high levels of maternal serumanti-polysaccharide antibody which may be transferred to the fetus inthe third trimester of pregnancy (30). Selection of appropriate carrierproteins is important for the development of polysaccharide-proteinvaccine formulations. For example, Haemophilus influenzae type b poly-or oligosaccharide conjugated to different protein carriers has variableimmunogenicity and elicits antibody with varying avidity (31, 32).Repeated immunization with the same carrier protein may also suppressimmune responses by competition for specific B cells (epitopicsuppression) or other mechanisms. This is of particular concern for thedevelopment of GBS vaccines since recently developed polyaccharide andoligosaccharide-protein conjugate vaccines against the bacteria H.influenzae, S. pneumoniae, and N. meningitidis all utilize a restrictednumber of carrier proteins (tetanus toxoid, CRM 197, diptheria toxoid),increasing the number of exposures to these carriers an individual islikely to receive. A “designer” vaccine, composed of a GBSpolysaccharide or oligosaccharide coupled to a GBS-specific carrierprotein, such as the novel GBS polypeptides, provided herein,particularly including Spb1, EmaC and EmaE, may be a preferablestrategy. The large size of certain of these novel GBS antigens may alsobe an advantage to traditional carrier proteins as increasing size isassociated with improved immunogenicity.

Example 3 EMA Homologs in Streptococci and Other Bacteria

As noted above, the GBS Ema proteins share segmental homology withcertain characterized proteins from other bacterial species, includingbacterial adhesion and invasion proteins. The segmental homolog is notedas in the range of 24-39%. In addition, the Ema proteins demonstratesome homology to one another. A comparison of the ema genes shows thatEmaA and EmaB are 47% homologous, however, due to the difference intheir predicted lengths it is necessary to insert gaps in the EmaAsequence in order to line them up. The two Ema proteins which are mostsimilar in structure, EmaC and EmaD share 48.7% amino acid homology toone another. EmaA/B, EmaC/D and EmaE are each .ltoreq.20% homologous toone another.

The ema sequences were used to search the unannotated microbial genomes(Eubacteria). The predicted Ema proteins were searched againsttranslations in all six frames (tblast x) of finished and unfinishedunannotated microbial genomes available at the web site of the NationalCenter for Biotechnology Information (NCBI). Segmental amino acidhomolog was identified.

EmaA has some segmental homolog with S. pneumoniae, E. faecalis, B.anthiacis and C. diptheriae. Ema B has some segmental homolog with B.anthracis. EmaE has segmental homology to S. pyogeizes and lesserhomology to B. anthracis.

Significant homology was identified between the GBS EmaC and EmaD andproteins in other bacterial species. EmaC has significant (55% identityover 149 amino acids) homology to a region of the S. pneumoniaechromosome and the S. pyogenes chromosome (47% identity over 150 aminoacids). Lesser segmental homology was found to E. faecalis, S. equi, andC. diptheriae. EmaD has strong segmental homology (66% over 184 aminoacids) to a region of the S. pneumoniae chromosome, and lesser segmentalhomology to C. diphtheriae and S. pyogenes.

We have identified two Ema homologs in S. pneumoniae. These S.pneumoniae homologs show homology to EmaC and EmaD and, like EmaC andEmaD, also demonstrate homology to fimbria-associated protein ofActinomyces. The encoding nucleic acid and predicted amino acid sequenceof the first S. pneumoniae EmaC/D homolog are provided in SEQ ID NOS: 24and 23, respectively. The genome region nucleic acid including the firsthomolog encoding sequence is provided in SEQ ID NO: 22. The nucleic acidand predicted amino acid sequence of the second S. pneumoniae EmaC/Dhomolog are provided in SEQ ID NOS: 27 and 26 respectively. The genomicregion nucleic acid of this second homolog is found in SEQ ID NO: 25. AnEmaC/D homolog has been identified in Enterococcus faecalis by searchand analysis. The E. faecalis EmaC/D homolog predicted amino acidsequence is provided in SEQ ID NO: 29. The nucleic acid sequenceencoding this E. faecalis Ema homolog is provided in SEQ ID NO: 30. Thenucleic acid sequence of E. faecalis which genomic region encodes theEmaC/D homolog is provided in SEQ ID NO: 28.

We have also identified an EmaD homolog in Corynebacterium diptheriae.The predicted amino acid sequence of the C. diptheriae EmaD homolog isprovided in SEQ ID NO: 32. C. diptheriae nucleic acid sequence whichencodes the homolog is found in SEQ ID NO: 33. The corresponding genomicregion sequence of C. diptheriae is provided in SEQ ID NO: 31.

A predicted EmaC/D homolog has been identified in S. pyogenes. Thepredicted partial amino acid sequence of this Ema homolog provided inSEQ ID NO: 37.

A region of amino acids TLLTCTPYMINS/THRLLVR/KG (SEQ ID NO: 34) is foundin GBS EmaC, GBS EmaD, in both the EmaC/D homologs of S. pneunoniae, andin the E. faecalis Ema homolog. A similar sequence TLVTCTPYGINTHRLLVTA(SEQ ID NO: 35) is also found in the C. diptheriae Ema homolog. The S.pyogenes predicted Ema homolog has a similar sequenceTLVTCTPYGVNTKRLLVRG (SEQ ID NO: 36) as well.

The following is a list of the references referred to in this Examplesection.

REFERENCES

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This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, the scope of the invention beingindicated by the appended Claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each ofwhich is incorporated herein by reference in its entirety.

1. An isolated antibody which specifically binds to streptococcalExtracellular Matrix Adhesion Protein A (EmaA) comprising the amino acidsequence set forth in SEQ ID NO:2 or a polypeptide comprising aminoacids 28-245 of SEQ ID NO:2, wherein the specific binding is to anepitope contained within the amino acid sequence set forth in SEQ IDNO:2 or a polypeptide comprising amino acids 28-245 of SEQ ID NO:2. 2.The antibody of claim 1, wherein said antibody is a monoclonal antibody.3. An immortal cell line that produces the monoclonal antibody of claim2.
 4. The antibody of claim 1 labeled with a detectable label.
 5. Theantibody of claim 4, wherein the label is selected from the groupconsisting of an enzyme, a chemical which fluoresces, and a radioactiveelement.
 6. A pharmaceutical composition comprising one or moreantibodies which specifically bind to streptococcal Extracellular MatrixAdhesion Protein A (EmaA), and a pharmaceutically acceptable carrier,wherein EmaA comprises the amino acid sequence set forth in SEQ ID NO:2or a polypeptide comprising amino acids 28-245 of SEQ ID NO:2, andwherein the specific binding is to an epitope contained within the aminoacid sequence set forth in SEQ ID NO:2 or a polypeptide comprising aminoacids 28-245 of SEQ ID NO:2.
 7. The pharmaceutical composition of claim6, further comprising at least one antibody to a protein selected fromthe group of Spb1 and Spb2, Rib, Lmb, C5a-ase and C protein alphaantigen.
 8. A method for treating infection with a bacterium expressingstreptococcal Extracellular Matrix Adhesion Protein A (EmaA) comprisingadministering a therapeutically effective dose of the pharmaceuticalcomposition of claim 6 to a subject in need thereof so that thesubject's infection is treated.
 9. A method for detecting the presenceof streptococcal Extracellular Matrix Adhesion Protein A (EmaA)comprising a. contacting a sample, wherein the presence or activity ofan EmaA streptococcal polypeptide is suspected, with the antibody ofclaim 1 under conditions that allow binding of the EmaA streptococcalpolypeptide and antibody; and b. detecting whether binding has occurredbetween the EmaA streptococcal polypeptide from the sample and theantibody; wherein the detection of binding indicates the presence oractivity of EmaA streptococcal polypeptide in the sample.